Gene content and coding diversity of growth hormone loci of apes

Gene content and coding diversity of growth hormone loci of apes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gene content and coding diversity of growth hormone loci of apes Rafael González-Álvarez, Irám Pablo Rodríguez-Sánchez, Hugo Alberto Barrera-Saldaña This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1844540/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Growth hormone (GH) locus experienced dramatic evolution in primates, becoming multigenic and diverse in anthropoids. Despite sequence information from vast number of primate species, it has remained unclear how the multigene family was favored feature. We sought to compare the structure and composition of the apes’ GH loci as a prerequisite to understanding their origin and possible evolutionary role. GH loci of chimpanzee, gorilla, and orangutan were sequenced from bacterial artificial chromosomes, while those of modern man, neanderthal, gibbon, and wild boar were retrieved from GenBank. Coding regions, regulatory elements, and repetitive sequences were identified and compared among species. GH loci of all analyzed species are flanked by the genes CD79B (5´) and ICAM-1 (3´). In man, neanderthal, and chimpanzee the loci are integrated by five genes almost indistinguishable, however while in the former two they render three different hormones, in the latter one four different proteins are derived. Gorilla exhibits six genes, gibbon seven, and orangutan four. The sequences of proximal promoters, enhancers, P-elements, and a locus control region (LCR) are highly conserved. The locus evolution might have implicated duplications of the ancestral pituitary GH-N gene and subsequent diversification of the copies, to lead to the placental single GH-V gene and to the multiple also placental CSH genes. chorionic somatomammotropins somatolactins prolactin platyrrhine cercopithecidae monkeys Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Growth hormones (GH), placental lactogens (PL), prolactins (PRL), prolactins 2 (PRL2), and somatolactins (SL) constitute in vertebrates a family of proteins within the large superfamily of class-1 helical cytokines [ 1 ]. All display a characteristic conserved amino-acid framework with four cysteine residues forming two disulfide bridges that stabilize their four-helical tertiary structure [ 2 , 3 , 4 ]. Even though there is considerable variation among members of this family in evolutionary rate across taxa and that distant members share only about 25–30% amino acid sequence identity, their conserved cysteine framework, the exon organization of their genes, and the binding properties of the members to their respective receptors, define them as a monophyletic family within the cytokine superfamily [ 1 ]. In primates, the GH locus experienced a dramatic evolution, resulting in the gene cluster coding at least three types of hormones: the pituitary one (GH-N), its placental variant (GH-V), and the chorionic somatomammotropin hormone (CSH, previously referred as placental lactogen), which appeared for the first time in Cercopithecidae . Despite the availability of considerable gene sequence information from a vast number of primate species, it has remained unclear how the gene family was formed, and different proposals have been discussed extensively over the years. A predominant hypothesis states that gene duplications of an ancestral version of the PRL gene gave rise to two branches, the PRL itself and the GH branch. While in mammals other than primates the former gave rise to placental lactogen ( PL ) and alike, in primates the GH branch rendered the GH-V gene and the CSH genes [ 5 , 6 , 7 ]. While most mammalian species have a single gene that encodes GH, in the superfamilies Platyrrhine (New World monkeys) and Cercopithecidae (Old World monkeys and apes, including humans) of primates, gene clusters emerged and diverged independently via dramatic events of gene duplication and conversion [ 8 , 9 , 10 ]. In fact, at the bottom of the primate evolutionary tree in prosimians, such as the slow loris, the GH and PRL proteins show a higher sequence similarity between them and with the GH of non-primate mammals, such as the wild boar, than with the GHs of higher primates. Thus, it is inferred that after the separation of the prosimian and higher primates’ lineages, the accelerated series of evolutionary events that characterize this gene family, began. This could be due to positive selection on the original gene, related to adaptive changes in the functioning of its encoded hormone, especially on its metabolic support to reproduction [ 11 , 12 ]. These events of gene duplications, gene conversions, and diversification of the new genes derived from the ancestral GH gene that happened in primates, constitute a dramatic evolutionary story that makes this locus an extraordinary case for molecular evolution studies. Their implications span from the emergence of spatial and temporal control of gene expression to a still non-resolved role of the multiple new encoded hormones in the endocrinology of pregnancy, and its possible implications on the survival of the human fetus [ 13 , 14 ]. GH performs its biological activity among species via the actions of the somatotrophic axis [ 15 ]. Human disorders, including reduced stature and delayed sexual maturity, can result when the normal actions of GH are disrupted [ 16 , 17 ]. Humans, as most higher anthropoids, are characterized by prolonged gestation and delayed rates of fetal maturation, with many anthropoid newborns having large brains relative to their body sizes. These features have been advanced as the basis for increased social complexity and cognitive capacity in said primates. The genetic basis of these characteristic anthropoid phenotypes is unknown; however, fetal development depends on access to maternal resources during pregnancy. Indeed, it has recently been shown that hemochorial placentation seen in anthropoids is associated with steeper brain-body allometry, faster prenatal brain growth, and slower prenatal body growth [ 18 ]. The placental variants of GH and the CSHs, have been implicated in the fetal acquisition of maternal resources during anthropoid pregnancy [ 19 ]. GH related genes that emerged in primates such as those in man ( CSH-L , CSH-A , GH-V , and CSH-B ) [ 8 ] and rhesus macaque ( GH-2, CSH-1, CSH-2, CSH-3 and, CSH-4 ) [ 20 ], and most likely in the rest of groups of primates, are transcribed in the placenta. Although largely uncharacterized, the hormones derived from these placenta-expressed genes have been implicated in diverse roles during pregnancy, from mediating trophoblast invasion [ 21 ] to regulating maternal resource availability for the developing fetus [ 22 ]. It should be noted that the expression of the elements of the GH locus is not limited to the pituitary gland or the placenta. Studies carried out since the last quarter of the past century have demonstrated the presence of GH in several extra-pituitary tissues such as neural, ocular, reproductive, immune, cardiovascular, muscular, dermal, and skeletal tissues [ 23 ]. For instance, the presence of GH in ocular structures such as retinal, choroidal, and corneal tissues, seems to function as an emergency mechanism against injuries. Because of this, GH can even be used as a biomarker to follow retinal neurodegeneration [ 24 ]. This is an intriguing case that reflects not only the need to further investigate new physiological functions of GH but also to unravel the surely complex mechanisms that regulate its extra-pituitary gene expression. Thus, the study of the evolutionary history of these genes clusters uniquely shared among anthropoids can illuminate important aspects of human pregnancy, fetal development, and further unknown physiological processes. Given that these physiological features are of recent acquisition in evolution, the putative contribution of the GH family must be reflected in its evolutionary history in primates. Therefore, to evaluate GH evolution in primates more systematically, we sought to examine and compare the composition and structure of GH loci in anthropoids’ species. Material And Methods Biological samples Bacterial Artificial Chromosomes (BACs) containing the GH locus from chimpanzee ( Pan troglodytes ), gorilla ( Gorilla gorilla ), and orangutan ( Pongo abelli ) were obtained from BACPAC Resource Center (Children’s Hospital Oakland Research Institute, Oakland, CA). The BAC’s GH-like gene sequences were amplified and cloned as previously described [ 25 , 26 ] while gene, intergenic, and flanking sequences were obtained by NGS with the technical assistance of Genome Quebec. Reads were assembled using Geneious prime software (v. 2021.0.1) by mapping with reference genomes available in the NCBI GenBank database. Sequences from human ( Homo sapiens ), Neanderthal ( Homo sapiens neanderthalensis ), gibbon ( Nomascus leucogenys ), and wild boar ( Sus scrofa ) were retrieved from the complete or partial respective genome sequences available in the GenBank (Table 1 ). Table 1 Sequences used in this study. Specie Accession number Human ( Homo sapiens ) J03071 Neanderthal ( Homo sapiens neanderthalensis ) GCA_000208225 Chimpanzee ( Pan troglodytes ) * Pending Gorilla ( Gorilla gorilla ) * Pending Orangutan ( Pongo abelii ) * OL672210 Gibbon ( Nomascus leucogenys ) CM016963.1 Wild boar ( Sus scrofa ) GCF_000003025 * Loci reported in this work. Genomic annotations Annotation of genetic elements (such as genes and their exons and introns) of the nucleotide sequences analyzed in this work was carried out using the software SnapGene 5.2. Identification of repetitive elements, predominantly present in intergenic regions of the GH locus , was made with the use of RepeatMasker 4.0.9 program [ 27 ] with default parameters (HMMER o NCBI/RMBLAST search methods). Regulatory elements were searched by similarity with the previously characterized ones [ 25 , 26 ], identifying response elements such as the Sp1 elements, the distal and proximal Pit-1 binding sites, the thyroid hormone response element (TRE), the initiator element (InrE), the cyclic AMP response element (CRE), and the TATA box, mainly. Phylogenetic analysis The sequences were aligned using the Clustal Omega program [ 28 ] followed by manual corrections in case of need. Protein sequences were derived by conceptual translation of the coding sequences. From coding sequences, a phylogenetic tree was built with MEGA 6.06 software [ 29 ] using the Neighbor-Joining (NJ) method; then a bootstrap test was done with 1,000 replicates [ 30 ]. The nucleotide sequences of the GH locus and flanking genes used in this study are listed in Table 1 . Wild boar ( Sus scrofa ) was used as the external group for the evolutionary analyses. Results Organization of the primate GH loci The GH locus in higher primates invariably contains the original pituitary or hypophyseal growth hormone gene ( GH-N ), its placental variant ( GH-V ) gene, and a variable number of chorionic somatomammotropin hormone ( CSH ) genes. As shown in Fig. 1 the loci of all species here investigated are flanked by the genes CD79B and ICAM-1 , including the one of the wild boar. The locus contains five genes in man, Neanderthal, and chimpanzee, while it harbors six genes in gorilla, four in orangutan, and seven in gibbon; as expected, the wild boar carries a single GH-N gene. It is noteworthy that GH-N and GH-V genes as well as the CSH-B gene are invariably present in all higher primates, and that the differences between their loci lie in the diversity of the CSH-A gene. In modern man, Neanderthal, orangutan, and gibbon the gene immediately following the GH-N , the CSH-L , carries a splicing mutation, becoming a pseudogene. Anatomy of the human GH locus The GH locus in man encompasses 75,316 bp. It harbors the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig. 2 ), 22 sense and 26 anti-sense Alu sequences (Table 2 ). Table 2 Repetitive elements found in the human, neanderthal, chimpanzee, gorilla and orangutan GH loci. Human Neanderthal Chimpanzee Gorilla Orangutan Locus length (bp) 75,316 75,287 84,237 98,181 63,107 Alu sense 22 28 15 29 18 Alu anti sense 26 28 15 23 18 Retrotasposon 0 0 2 0 1 Dissection of the GH locus in the Neanderthal man This locus encompasses 75,287 bp and contains the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig. 2 ), as well as 28 sense and an equal number of anti-senses Alu sequences (Table 2 ). Examination of the chimpanzee’s GH locus The locus encompasses 84,237 bp and harbors the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig. 2 ), as well as 30 Alu sequences, half sense and half antisense, plus one retrotrasposon (Table 2 ). Analysis ohe GH locus in gorilla . This is the largest locus , spanning 98,181 bp, and enclose Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig. 2 ), as well as 29 sense and 23 anti-sense Alu sequences (Table 2 ). Features of the orangutan’s GH locus This locus is the shortest, constituted of only 63,107 bp, which otherwise also accomodates the Sp-1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA and InrE regulatory elements (Fig. 2 ), as well as 18 sense Alu sequences, 18 anti-sense Alu sequences and 1 retrotrasposon (Table 2 ). Description of the gibbon’s GH locus The locus still as a draft, thus we did not include it in the comparison. Phylogenetic analys es A phylogenetic tree was constructed based on the cDNA sequences of the GH / CSH genes using the Neighbor Joining (NJ) method relying on one sequence as outgroup (wild boar). As depicted in Fig. 3 , its branches show well defined clades, such as GH-N , GH-V , CSH-A / B , and the pseudogene CSH-L . Interestingly, the gibbon GH locus shows two GH-V genes (Figs. 1 and 2 ), but they make clade with the GH-N genes, which suggests that this deviation of the patterns of gene composition for the loci of all species of primates analyzed so far, may be due to an annotation mistake, since this genome still as draft. The presence of two GH-N or GH-V genes is very unprovable. Regulatory elements As described previously [ 25 , 26 ], regulatory elements in the promoters, enhancers, and a locus control region (LCR), which is outside the GH locus limits, were identified by sequence similarity. The promoter regions are conserved in the different genes in all species. The Sp1 element and the TATA box are completely conserved, while the distal Pit-1 is highly conserved, being identical in the GH-N and the GH-V genes but showing only one nucleotide substitution in the CSH genes. On the other hand, substitutions are observed in the proximal Pit-1 binding site in the GH-V genes. Likewise, the TRE shows considerable variation among the genes. On the other hand, the CRE is well conserved, having only two substitutions in the orangutan GH-N gene, one in the gorilla CSH-B gene, and one in each of the orangutan GH-V, CSH-L , and CSH-B genes. The InrE shows substitutions mainly in the CSH and the GH-V genes. The placental enhancers are located downstream from the CSH genes. They are well conserved and display all the typical organization in four domains (DF-1 to DF-4). only a few substitutions are observed in its DF-1 and DF-2 domains. A substitution is present in DF-1 (position 16) in the CSH-B genes’ enhancers (except for the orangutan). These enhancers also show deletions at the upstream region of the DF-2 domain in man, neanderthal, chimpanzee, and gorilla. Moreover, a substitution in position 65 is also present in these enhancers. With respect to the domains DF-3 and DF-4, they show a few mutations, particularly a substitution in DF-3 in the position 228. Other mutations are also present in the primate CSH enhancer sequences, like the ones observed at positions 112 and 134. However, these mutations are not inside these enhancer’s domains. Gene content and diversity of encoded proteins The genes on the apes’ GH loci code for three distinctive types of proteins: the pituitary GH-N, the placental GH-V, and CSHs. The GH-N and GH-V genes are constants, code for well differentiated proteins, and there is only one copy of each in each locus . The protein encoded by the GH-N gene is the most well conserved of all proteins coded by the loci of all the apes, being identical in human, neanderthal, chimpanzee, gorilla, and orangutan, although surprisingly it has 4–5 changes in gibbon (see blue boxes in table 3). The protein encoded by the GH-V gene is also well conserved showing 0, 1, 4, 11 and 14 amino acid changes in neanderthal, chimpanzee, gorilla, orangutan, and gibbon, respectively (see pink boxes in table 3). In contrast to the GH genes, the CSH genes differ in number and in their capacity to render protein diversity, since the encoded proteins in a few of these genes are in some cases identical (such as in the case of the two functional genes of the human locus ), and in other apes the gene following the GH-N , the so called CSH-L , no longer contributes protein since it became a pseudogene. Also, from the point of view of diversity, the proteins encoded by the CSH genes are more diverse, as it is shown in the orange boxes in table 3. While CSH genes in number vary from one to four, the number of different encoded proteins does not follow the gene count. For example, in the human there are CSH genes: CSH-L , CSH-A, and CSH-B , but with the first being a pseudogene and the two-remaining encoding the same mature protein (see red box in table 3), the gene count of three gets reduced to a protein count of one. Thus, five GH-CSH human genes produces three proteins (one GH-N, one GH-V, and one CSH). A similar pattern is seen in the neanderthal. Since in the chimpanzee, the CSH-A1 gene produces the same mature protein that does the CSH-A2 (red box in table 3), its GH locus renders four proteins: one GH-N, one GH-V, and two different CSHs. Then, for the gorilla CSH genes, three different mature proteins derive, since its CSH-A2 gene produces the same protein than its CSH-A3 gene (red box in table 3). Thus, the gorilla GH locus produces five proteins: one GH-N, one GH-V, and three CSHs. Orangutan GH locus has two CSH genes, but one is a pseudogene. Thus, the orangutan GH locus produces three proteins: one GH-N, one GH-V, and one CSH. Finally, the gibbon has four CSH genes being one of them also a pseudogene and the rest do not show obvious mutations that could suggest failure in their expression. Therefore, the gibbon GH locus apparently produces six proteins: two GH-N, one GH-V, and three CSHs. A summary of these gene counts versus potentiality to render diverse proteins is depicted in table 3. Discussion Understanding the evolutionary forces that shaped the multigene family of GH and PRL in mammals have been a subject of great interest. In this study, we have investigated the gene composition and arrangement of anthropoidean GH loci . To explain their present-day anatomy, we have invoked gene duplications and specializations events, which could have giving rise to the GH family in higher primates. Our phylogenetic analysis approach is broader, covering the genomic architecture of the loci in these species, the inspection of the synonymous (dS) versus nonsynonymous (dN) substitution ratios in their nucleotide sequences [ 11 , 12 , 13 ], and the conceptual translation of their genes to unravel their coding diversity potential. The anatomy of the GH loci in primates as depicted in Fig. 1 shows that the organization of their gene members in the following general order: GH-N , CSH-A (variable number), GH-V , and CSH-B . The loci always begin with GH-N , then follows CSHs (including the CSH-L) , GH-V , and finally one more CSH gene. All these loci , including all known non-primate loci , are flanked by the CD79B gene at the 5’ end by the ICAM-1 gene at the 3’ end. The GH loci of the analyzed species of apes have several repetitive sequences of the Alu family in an average of one every 1,000 pb. It is well known that this type of repetitive sequences favors the non-homologous recombination events that originate multigene families. Given that in primates the flanking genes do not change much, GH-N always is the first gene, and the presence of numerous Alu sequences, it can be inferred that duplications events and nucleotide changes that originated this family occurred only within the GH loci. The way in how it happened might well resulted from the alternative evolutionary routes proposed in Fig. 4 . The phylogenetic analysis shown in the tree of Fig. 3 displays four well defined and well supported clades by the bootstrap analysis, being from top to bottom GH-N , GH-V , CSH , and non-primate GH (out-group). It indicates that GH-N is the orthologous gene, while GH-V and the CSHs are the paralogous ones. This analysis supports the evolutionary pathway depicted in Fig. 4 , with two possible routes. In the first route, the ancestral GH-N gene duplicated to give rise to GH-N like genes. Then, these experienced a series of mutations that gave rise to the placental genes ( GH-V and CSHs ), and later to the pseudogenization of a CSH gene. In a variation of this route, the ancestral GH-N duplicated in one pre- GH-V gene and some pre- CSH genes; then a second round of duplication only acting at the pre- CSH genes gave rise to the various of CSH genes. Likewise, further mutations in the pre- GH-V resulted in the GH-V . Finally, a splicing mutation converted the CSH-L gene in a pseudogene. Another striking feature of the analyzed loci is the diversity of their encoded hormones. We found that CSH genes quantity does not match with the CSH mature protein diversity that they encode. Human and neanderthal have three CSH genes and they encode only one CSH mature protein. Chimpanzee has three CSH genes and they encode two CSH mature proteins. Gorilla has four CSH genes and they encode three CSH mature proteins. Orangutan has two CSH genes and they encode one CSH mature protein. Finally, the gibbon has four CSH genes and they encode three CSH mature proteins (table 3). This fact opens two interesting questions, such as: 1) why two genes encode the same protein? And 2) do they produce the same protein quantities? First question could be answered by the phylogenetic analysis performed previously [ 9 – 13 , 20 , 23 , 25 , 26 ]. They indicate that the evolutionary forces that underling the GH locus in higher primates is the purifying selection (dN < dS), this force being typical of functional genes. Although we lack information to solve the second question in all apes, we explored it in human and found that they produce similar amounts [ 31 ]. Although this could be an answer to the need to produce surprisingly high amounts of their encoded identical proteins, reaching a couple of grams at the end of pregnancy [ 32 ] to fully answer these and other related questions, expression profiles in the apes’ hypophysis and placenta might be needed. Declarations Acknowledgements The authors wish to express their gratitude to Ana Karen Sánchez and Abigail Torres Cerda for their valuable technical assistance with bioinformatic analyses. They also acknowledge the support of the Mexico’s National System of Researchers (SNI) and of their respective academic institutions. Competing interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. References Huising, M. O., C. P. Kruiswijk and G. Flik, (2006) Phylogeny and evolution of class-I helical cytokines. J Endocrinol 189: 1–25. Pérez-Sánchez, J., J. A. Calduch-Giner, M. Mingarro, S. Vega-Rubín de Celis, P. Gómez-Requeni et al. , (2002) Overview of Fish Growth Hormone Family. New Insights in Genomic Organization and Heterogeneity of Growth Hormone Receptors. Fish Physiology and Biochemistry 27: 243–258. Power, D. M., (2005) Developmental ontogeny of prolactin and its receptor in fish. Gen Comp Endocrinol 142: 25–33. Whittington, C. M., and A. B. Wilson, (2013) The role of prolactin in fish reproduction. Gen Comp Endocrinol 191: 123–136. Bole-Feysot, C., V. Goffin, M. Edery, N. Binart and P. A. Kelly, (1998) Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19: 225–268. Forsyth, I. A., and M. Wallis, (2002) Growth hormone and prolactin–molecular and functional evolution. J Mammary Gland Biol Neoplasia 7: 291–312. Kawauchi, H., and S. A. Sower, (2006) The dawn and evolution of hormones in the adenohypophysis. Gen Comp Endocrinol 148: 3–14. Chen, E. Y., Y. C. Liao, D. H. Smith, H. A. Barrera-Saldana, R. E. Gelinas et al. , (1989) The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 4: 479–497. Wallis, O. C., and M. Wallis, (2002) Characterisation of the GH gene cluster in a new-world monkey, the marmoset (Callithrix jacchus). J Mol Endocrinol 29: 89–97. Revol De Mendoza, A., D. Esquivel Escobedo, I. Martinez Davila and H. Saldana, (2004) Expansion and divergence of the GH locus between spider monkey and chimpanzee. Gene 336: 185–193. Wallis, O. C., Y. P. Zhang and M. Wallis, (2001) Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change. J Mol Endocrinol 26: 249–258. Wallis, O. C., A. O. Mac-Kwashie, G. Makri and M. Wallis, (2005) Molecular evolution of prolactin in primates. J Mol Evol 60: 606–614. Wallis, O. C., and M. Wallis, (2006) Evolution of growth hormone in primates: the GH gene clusters of the New World monkeys marmoset (Callithrix jacchus) and white-fronted capuchin (Cebus albifrons). J Mol Evol 63: 591–601. Papper, Z., N. M. Jameson, R. Romero, A. L. Weckle, P. Mittal et al. , (2009) Ancient origin of placental expression in the growth hormone genes of anthropoid primates. Proc Natl Acad Sci U S A 106: 17083–17088. Gluckman, P. D., and C. S. Pinal, (2002) Maternal-placental-fetal interactions in the endocrine regulation of fetal growth: role of somatotrophic axes. Endocrine 19: 81–89. Dattani, M., and M. Preece, (2004) Growth hormone deficiency and related disorders: insights into causation, diagnosis, and treatment. Lancet 363: 1977–1987. Zhou, Y., B. C. Xu, H. G. Maheshwari, L. He, M. Reed et al. , (1997) A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci U S A 94: 13215–13220. Elliot, M. G., and B. J. Crespi, (2008) Placental invasiveness and brain-body allometry in eutherian mammals. J Evol Biol 21: 1763–1778. Haig, D., (2008) Placental growth hormone-related proteins and prolactin-related proteins. Placenta 29 Suppl A: S36-41. Gonzalez Alvarez, R., A. Revol de Mendoza, D. Esquivel Escobedo, G. Corrales Felix, I. Rodriguez Sanchez et al. , (2006) Growth hormone locus expands and diverges after the separation of New and Old World Monkeys. Gene 380: 38–45. Lacroix, M. C., J. Guibourdenche, T. Fournier, I. Laurendeau, A. Igout et al. , (2005) Stimulation of human trophoblast invasion by placental growth hormone. Endocrinology 146: 2434–2444. Fleenor, D., J. Oden, P. A. Kelly, S. Mohan, S. Alliouachene et al. , (2005) Roles of the lactogens and somatogens in perinatal and postnatal metabolism and growth: studies of a novel mouse model combining lactogen resistance and growth hormone deficiency. Endocrinology 146: 103–112. Pérez-Ibave, D. C., I. P. Rodríguez-Sánchez, L. Garza-Rodríguez Mde and H. A. Barrera-Saldaña, (2014) Extrapituitary growth hormone synthesis in humans. Growth Horm IGF Res 24: 47–53. Pérez-Ibave, D. C., I. P. Rodríguez-Sánchez, M. L. Garza-Rodríguez, A. A. Pérez-Maya, M. Luna et al. , (2018) Expression of growth hormone gene in the baboon eye. Exp Eye Res 169: 157–169. Pérez-Maya, A. A., I. P. Rodríguez-Sánchez, P. de Jong, M. Wallis and H. A. Barrera-Saldaña, (2012) The chimpanzee GH locus: composition, organization, and evolution. Mamm Genome 23: 387–398. Pérez-Maya, A. A., M. Wallis and H. A. Barrera-Saldaña, (2016) Structure and evolution of the gorilla and orangutan growth hormone loci. Mamm Genome 27: 511–523. Smit, A., Hubley, R & Green P, (2013–2015) RepeatMasker Open-4.0, pp. Chenna, R., H. Sugawara, T. Koike, R. Lopez, T. J. Gibson et al. , (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31: 3497–3500. Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar, (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 30: 2725–2729. Saitou, N., and M. Nei, (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425. Martinez-Rodriguez, H. G., Guerra-Rodriguez, N. E., Iturbe-Cantu, M. A., Martinez-Torres, A., & Barrera-Saldaña, H. A. (1997). Expression of human placental lactogen and variant growth hormone genes in placentas. Arch Med Res, 28(4), 507–512. Barrera-Saldaña HA, Seeburg PH, Saunders GF (1983) Two structurally different genes produce the same secreted human placental lactogen hormone. The Journal of biological chemistry, 258(6):3787–93. table Table 3 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table3.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1844540","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":120093257,"identity":"71a292a0-e64a-45b9-ae54-b8f03ad24509","order_by":0,"name":"Rafael González-Álvarez","email":"","orcid":"","institution":"Instituto Tecnológico de los Altos de Jalisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"González-Álvarez","suffix":""},{"id":120093258,"identity":"b0e19e72-b8ff-4c83-8481-e40434399790","order_by":1,"name":"Irám Pablo Rodríguez-Sánchez","email":"","orcid":"","institution":"Universidad Autónoma de Nuevo León","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Irám","middleName":"Pablo","lastName":"Rodríguez-Sánchez","suffix":""},{"id":120093259,"identity":"8ed5876e-6ee1-49bf-8dc4-514acf50bfd3","order_by":2,"name":"Hugo Alberto Barrera-Saldaña","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACAwYeKIu9AcS1IEULzwEQV4IULRIJYJKwFnP2swc/V+6xyeef+fzqhh8FEgz87d0JeLVY9uQlS555lmY543ZO2c0eoMMkzpzdgN9hB3IMJBsOHDZguJ2TdoMHqMVAIpeAlvNvjH82HPhvIH/zTNrNP0RpuZFjBrTlgIHBDfZjt4mz5ca7NMuGA8kGhmdy2G7LGEjwEPbL+dzDNxsO2BnIHT/+7OabPzZy/O29+LUgAR4DMEmschBgf0CK6lEwCkbBKBhBAADmEErrtdE3RwAAAABJRU5ErkJggg==","orcid":"","institution":"Vitagénesis, SA de CV/Innbiogem, SC. Monterrey","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"Alberto","lastName":"Barrera-Saldaña","suffix":""}],"badges":[],"createdAt":"2022-07-10 21:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1844540/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1844540/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24048490,"identity":"6efe61ae-db46-44ea-85d8-d5df3c01a82a","added_by":"auto","created_at":"2022-07-19 16:34:56","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe hominids’ \u003cem\u003eGH\u003c/em\u003e \u003cem\u003eloci\u003c/em\u003e\u003c/strong\u003e. The genomic organization of the GH \u003cem\u003eloci\u003c/em\u003e of man (\u003cem\u003eHomo sapiens\u003c/em\u003e), Neanderthal (\u003cem\u003eHomo sapiens neanderthalensis\u003c/em\u003e), chimpanzee (\u003cem\u003ePan troglodytes\u003c/em\u003e), gorilla (\u003cem\u003eGorilla gorilla\u003c/em\u003e), orangutan (\u003cem\u003ePongo abelii\u003c/em\u003e), gibbon (\u003cem\u003eNomascus leucogenys\u003c/em\u003e), and wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) are schematized. The white arrows indicate the genes \u003cem\u003eCD79B\u003c/em\u003e and \u003cem\u003eICAM-1\u003c/em\u003e immediately flanking the \u003cem\u003eGH\u003c/em\u003e locus in all the assembly’s sequences reported in the present work. The direction of the\u003cem\u003e arrow\u003c/em\u003e indicates the direction of the genes’ transcription. Continuous lines indicated complete sequences, while discontinuous ones represent sequences that are missing.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/13e8834e3a4515afca5ea3fa.jpg"},{"id":24047662,"identity":"1085f2cf-e9d9-4450-9520-86bf6bc0877f","added_by":"auto","created_at":"2022-07-19 16:29:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApe’s \u003cem\u003eGH loci\u003c/em\u003e. \u003c/strong\u003eThe genomic organization of the GH \u003cem\u003eloci\u003c/em\u003e of man (\u003cem\u003eHomo sapiens\u003c/em\u003e), Neanderthal (\u003cem\u003eHomo sapiens neanderthalensis\u003c/em\u003e), chimpanzee (\u003cem\u003ePan troglodytes\u003c/em\u003e), gorilla (\u003cem\u003eGorilla gorilla\u003c/em\u003e) and orangutan (\u003cem\u003ePongo abelii\u003c/em\u003e) is shown. The blue arrows indicate the \u003cem\u003eCD79B\u003c/em\u003e and \u003cem\u003eICAM-1\u003c/em\u003e genes, while purple ones represent the \u003cem\u003eGH\u003c/em\u003e genes. The direction of the arrow indicates the direction of the genes’ transcription. Color lines indicate the regulatory and repetitive elements.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/4b0af3559e018759e4c3751e.jpg"},{"id":24048491,"identity":"dd0637b0-29c9-4dae-b5bc-aa4611c943e8","added_by":"auto","created_at":"2022-07-19 16:34:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree\u003c/strong\u003e. Neighbor-joining relationships were made in MEGA software based on the cDNA sequence of the \u003cem\u003eGH\u003c/em\u003e/\u003cem\u003eCSH\u003c/em\u003e genes of human, Neanderthal, chimpanzee, gorilla, orangutan, and gibbon. For the outgroup, to locate the root of the tree, sequence of wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) was used. Bootstrap percentages by 1,000 replications are shown on the branches.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/b3e30bcafa4f391894a967a1.jpg"},{"id":24047658,"identity":"240f9946-69c4-4047-b555-9f8ab52cef66","added_by":"auto","created_at":"2022-07-19 16:29:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo possible scenarios that gave rise the modern GH \u003cem\u003eloci\u003c/em\u003e in apes.\u003c/strong\u003e First scenario considers one duplication event followed by a mutations/specializations event. Second scenario consider two duplication, then mutations/specializations event and finally a second round of duplications events.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/89aad99d4c8a65338ec6ffe1.jpg"},{"id":27330954,"identity":"5b3d1f5e-7b70-47cc-b790-b458a98f3bc5","added_by":"auto","created_at":"2022-10-04 15:59:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":618590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/7405fc6d-3414-4e87-bf8a-7f36a1e5c7db.pdf"},{"id":24047660,"identity":"8d1871d9-8707-4309-837e-578e85ef0c8e","added_by":"auto","created_at":"2022-07-19 16:29:56","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45899,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1844540/v1/973f0271c59add3e264c0b1c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gene content and coding diversity of growth hormone loci of apes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrowth hormones (GH), placental lactogens (PL), prolactins (PRL), prolactins 2 (PRL2), and somatolactins (SL) constitute in vertebrates a family of proteins within the large superfamily of class-1 helical cytokines [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. All display a characteristic conserved amino-acid framework with four cysteine residues forming two disulfide bridges that stabilize their four-helical tertiary structure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Even though there is considerable variation among members of this family in evolutionary rate across taxa and that distant members share only about 25\u0026ndash;30% amino acid sequence identity, their conserved cysteine framework, the exon organization of their genes, and the binding properties of the members to their respective receptors, define them as a monophyletic family within the cytokine superfamily [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In primates, the \u003cem\u003eGH locus\u003c/em\u003e experienced a dramatic evolution, resulting in the gene cluster coding at least three types of hormones: the pituitary one (GH-N), its placental variant (GH-V), and the chorionic somatomammotropin hormone (CSH, previously referred as placental lactogen), which appeared for the first time in \u003cem\u003eCercopithecidae\u003c/em\u003e. Despite the availability of considerable gene sequence information from a vast number of primate species, it has remained unclear how the gene family was formed, and different proposals have been discussed extensively over the years. A predominant hypothesis states that gene duplications of an ancestral version of the \u003cem\u003ePRL\u003c/em\u003e gene gave rise to two branches, the \u003cem\u003ePRL\u003c/em\u003e itself and the \u003cem\u003eGH\u003c/em\u003e branch. While in mammals other than primates the former gave rise to placental lactogen (\u003cem\u003ePL\u003c/em\u003e) and alike, in primates the \u003cem\u003eGH\u003c/em\u003e branch rendered the \u003cem\u003eGH-V\u003c/em\u003e gene and the \u003cem\u003eCSH\u003c/em\u003e genes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile most mammalian species have a single gene that encodes GH, in the superfamilies \u003cem\u003ePlatyrrhine\u003c/em\u003e (New World monkeys) and \u003cem\u003eCercopithecidae\u003c/em\u003e (Old World monkeys and apes, including humans) of primates, gene clusters emerged and diverged independently via dramatic events of gene duplication and conversion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In fact, at the bottom of the primate evolutionary tree in prosimians, such as the slow loris, the GH and PRL proteins show a higher sequence similarity between them and with the GH of non-primate mammals, such as the wild boar, than with the GHs of higher primates. Thus, it is inferred that after the separation of the prosimian and higher primates\u0026rsquo; lineages, the accelerated series of evolutionary events that characterize this gene family, began. This could be due to positive selection on the original gene, related to adaptive changes in the functioning of its encoded hormone, especially on its metabolic support to reproduction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese events of gene duplications, gene conversions, and diversification of the new genes derived from the ancestral \u003cem\u003eGH\u003c/em\u003e gene that happened in primates, constitute a dramatic evolutionary story that makes this locus an extraordinary case for molecular evolution studies. Their implications span from the emergence of spatial and temporal control of gene expression to a still non-resolved role of the multiple new encoded hormones in the endocrinology of pregnancy, and its possible implications on the survival of the human fetus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGH performs its biological activity among species via the actions of the somatotrophic axis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Human disorders, including reduced stature and delayed sexual maturity, can result when the normal actions of GH are disrupted [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Humans, as most higher anthropoids, are characterized by prolonged gestation and delayed rates of fetal maturation, with many anthropoid newborns having large brains relative to their body sizes. These features have been advanced as the basis for increased social complexity and cognitive capacity in said primates. The genetic basis of these characteristic anthropoid phenotypes is unknown; however, fetal development depends on access to maternal resources during pregnancy. Indeed, it has recently been shown that hemochorial placentation seen in anthropoids is associated with steeper brain-body allometry, faster prenatal brain growth, and slower prenatal body growth [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The placental variants of GH and the CSHs, have been implicated in the fetal acquisition of maternal resources during anthropoid pregnancy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eGH\u003c/em\u003e related genes that emerged in primates such as those in man (\u003cem\u003eCSH-L\u003c/em\u003e, \u003cem\u003eCSH-A\u003c/em\u003e, \u003cem\u003eGH-V\u003c/em\u003e, and \u003cem\u003eCSH-B\u003c/em\u003e) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and rhesus macaque (\u003cem\u003eGH-2, CSH-1, CSH-2, CSH-3\u003c/em\u003e and, \u003cem\u003eCSH-4\u003c/em\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and most likely in the rest of groups of primates, are transcribed in the placenta. Although largely uncharacterized, the hormones derived from these placenta-expressed genes have been implicated in diverse roles during pregnancy, from mediating trophoblast invasion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] to regulating maternal resource availability for the developing fetus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt should be noted that the expression of the elements of the \u003cem\u003eGH\u003c/em\u003e locus is not limited to the pituitary gland or the placenta. Studies carried out since the last quarter of the past century have demonstrated the presence of GH in several extra-pituitary tissues such as neural, ocular, reproductive, immune, cardiovascular, muscular, dermal, and skeletal tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For instance, the presence of GH in ocular structures such as retinal, choroidal, and corneal tissues, seems to function as an emergency mechanism against injuries. Because of this, GH can even be used as a biomarker to follow retinal neurodegeneration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This is an intriguing case that reflects not only the need to further investigate new physiological functions of GH but also to unravel the surely complex mechanisms that regulate its extra-pituitary gene expression.\u003c/p\u003e \u003cp\u003eThus, the study of the evolutionary history of these genes clusters uniquely shared among anthropoids can illuminate important aspects of human pregnancy, fetal development, and further unknown physiological processes. Given that these physiological features are of recent acquisition in evolution, the putative contribution of the GH family must be reflected in its evolutionary history in primates. Therefore, to evaluate GH evolution in primates more systematically, we sought to examine and compare the composition and structure of \u003cem\u003eGH\u003c/em\u003e loci in anthropoids\u0026rsquo; species.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiological samples\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBacterial Artificial Chromosomes (BACs) containing the \u003cem\u003eGH locus\u003c/em\u003e from chimpanzee (\u003cem\u003ePan troglodytes\u003c/em\u003e), gorilla (\u003cem\u003eGorilla gorilla\u003c/em\u003e), and orangutan (\u003cem\u003ePongo abelli\u003c/em\u003e) were obtained from BACPAC Resource Center (Children\u0026rsquo;s Hospital Oakland Research Institute, Oakland, CA). The BAC\u0026rsquo;s GH-like gene sequences were amplified and cloned as previously described [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] while gene, intergenic, and flanking sequences were obtained by NGS with the technical assistance of Genome Quebec. Reads were assembled using Geneious prime software (v. 2021.0.1) by mapping with reference genomes available in the NCBI GenBank database. Sequences from human (\u003cem\u003eHomo sapiens\u003c/em\u003e), Neanderthal (\u003cem\u003eHomo sapiens neanderthalensis\u003c/em\u003e), gibbon (\u003cem\u003eNomascus leucogenys\u003c/em\u003e), and wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) were retrieved from the complete or partial respective genome sequences available in the GenBank (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSequences used in this study.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecie\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAccession number\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman (\u003cem\u003eHomo sapiens\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJ03071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeanderthal (\u003cem\u003eHomo sapiens neanderthalensis\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGCA_000208225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChimpanzee (\u003cem\u003ePan troglodytes\u003c/em\u003e) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePending\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGorilla (\u003cem\u003eGorilla gorilla\u003c/em\u003e) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePending\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOrangutan (\u003cem\u003ePongo abelii\u003c/em\u003e) *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOL672210\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGibbon (\u003cem\u003eNomascus leucogenys\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCM016963.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWild boar (\u003cem\u003eSus scrofa\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGCF_000003025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e* \u003cem\u003eLoci\u003c/em\u003e reported in this work.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGenomic annotations\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eAnnotation of genetic elements (such as genes and their exons and introns) of the nucleotide sequences analyzed in this work was carried out using the software SnapGene 5.2. Identification of repetitive elements, predominantly present in intergenic regions of the \u003cem\u003eGH locus\u003c/em\u003e, was made with the use of RepeatMasker 4.0.9 program [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] with default parameters (HMMER o NCBI/RMBLAST search methods). Regulatory elements were searched by similarity with the previously characterized ones [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], identifying response elements such as the Sp1 elements, the distal and proximal Pit-1 binding sites, the thyroid hormone response element (TRE), the initiator element (InrE), the cyclic AMP response element (CRE), and the TATA box, mainly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences were aligned using the Clustal Omega program [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] followed by manual corrections in case of need. Protein sequences were derived by conceptual translation of the coding sequences. From coding sequences, a phylogenetic tree was built with MEGA 6.06 software [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] using the Neighbor-Joining (NJ) method; then a bootstrap test was done with 1,000 replicates [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The nucleotide sequences of the \u003cem\u003eGH locus\u003c/em\u003e and flanking genes used in this study are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) was used as the external group for the evolutionary analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOrganization of the primate\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH loci\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eGH\u003c/em\u003e locus in higher primates invariably contains the original pituitary or hypophyseal growth hormone gene (\u003cem\u003eGH-N\u003c/em\u003e), its placental variant (\u003cem\u003eGH-V\u003c/em\u003e) gene, and a variable number of chorionic somatomammotropin hormone (\u003cem\u003eCSH\u003c/em\u003e) genes. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e the \u003cem\u003eloci\u003c/em\u003e of all species here investigated are flanked by the genes \u003cem\u003eCD79B\u003c/em\u003e and \u003cem\u003eICAM-1\u003c/em\u003e, including the one of the wild boar. The locus contains five genes in man, Neanderthal, and chimpanzee, while it harbors six genes in gorilla, four in orangutan, and seven in gibbon; as expected, the wild boar carries a single \u003cem\u003eGH-N\u003c/em\u003e gene. It is noteworthy that \u003cem\u003eGH-N\u003c/em\u003e and \u003cem\u003eGH-V\u003c/em\u003e genes as well as the \u003cem\u003eCSH-B\u003c/em\u003e gene are invariably present in all higher primates, and that the differences between their \u003cem\u003eloci\u003c/em\u003e lie in the diversity of the \u003cem\u003eCSH-A\u003c/em\u003e gene. In modern man, Neanderthal, orangutan, and gibbon the gene immediately following the \u003cem\u003eGH-N\u003c/em\u003e, the \u003cem\u003eCSH-L\u003c/em\u003e, carries a splicing mutation, becoming a pseudogene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnatomy of the human\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eGH locus\u003c/em\u003e in man encompasses 75,316 bp. It harbors the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), 22 sense and 26 anti-sense \u003cem\u003eAlu\u003c/em\u003e sequences (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRepetitive elements found in the human, neanderthal, chimpanzee, gorilla and orangutan GH loci.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNeanderthal\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChimpanzee\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGorilla\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrangutan\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLocus length (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75,316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75,287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e84,237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98,181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63,107\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAlu\u003c/em\u003e sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAlu\u003c/em\u003e anti sense\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRetrotasposon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDissection of the\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e \u003cstrong\u003ein the Neanderthal man\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis \u003cem\u003elocus\u003c/em\u003e encompasses 75,287 bp and contains the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), as well as 28 sense and an equal number of anti-senses \u003cem\u003eAlu\u003c/em\u003e sequences (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExamination of the chimpanzee\u0026rsquo;s\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe locus encompasses 84,237 bp and harbors the Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), as well as 30 \u003cem\u003eAlu\u003c/em\u003e sequences, half sense and half antisense, plus one retrotrasposon (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis ohe\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e \u003cstrong\u003ein gorilla\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThis is the largest \u003cem\u003elocus\u003c/em\u003e, spanning 98,181 bp, and enclose Sp1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA, and InrE regulatory elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), as well as 29 sense and 23 anti-sense \u003cem\u003eAlu\u003c/em\u003e sequences (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeatures of the orangutan\u0026rsquo;s\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThis \u003cem\u003elocus\u003c/em\u003e is the shortest, constituted of only 63,107 bp, which otherwise also accomodates the Sp-1, distal Pit-1, CRE, proximal Pit-1, TRE, TATA and InrE regulatory elements (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), as well as 18 sense \u003cem\u003eAlu\u003c/em\u003e sequences, 18 anti-sense \u003cem\u003eAlu\u003c/em\u003e sequences and 1 retrotrasposon (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDescription of the gibbon\u0026rsquo;s\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eGH locus\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003elocus\u003c/em\u003e still as a draft, thus we did not include it in the comparison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analys\u003c/strong\u003e\u003cstrong\u003ees\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA phylogenetic tree was constructed based on the cDNA sequences of the \u003cem\u003eGH\u003c/em\u003e/\u003cem\u003eCSH\u003c/em\u003e genes using the Neighbor Joining (NJ) method relying on one sequence as outgroup (wild boar). As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, its branches show well defined clades, such as \u003cem\u003eGH-N\u003c/em\u003e, \u003cem\u003eGH-V\u003c/em\u003e, \u003cem\u003eCSH-A\u003c/em\u003e/\u003cem\u003eB\u003c/em\u003e, and the pseudogene \u003cem\u003eCSH-L\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eInterestingly, the gibbon \u003cem\u003eGH\u003c/em\u003e locus shows two \u003cem\u003eGH-V\u003c/em\u003e genes (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), but they make clade with the \u003cem\u003eGH-N\u003c/em\u003e genes, which suggests that this deviation of the patterns of gene composition for the \u003cem\u003eloci\u003c/em\u003e of all species of primates analyzed so far, may be due to an annotation mistake, since this genome still as draft. The presence of two \u003cem\u003eGH-N\u003c/em\u003e or \u003cem\u003eGH-V\u003c/em\u003e genes is very unprovable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegulatory elements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs described previously [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e], regulatory elements in the promoters, enhancers, and a \u003cem\u003elocus\u003c/em\u003e control region (LCR), which is outside the \u003cem\u003eGH locus\u003c/em\u003e limits, were identified by sequence similarity. The promoter regions are conserved in the different genes in all species. The Sp1 element and the TATA box are completely conserved, while the distal Pit-1 is highly conserved, being identical in the \u003cem\u003eGH-N\u003c/em\u003e and the \u003cem\u003eGH-V\u003c/em\u003e genes but showing only one nucleotide substitution in the \u003cem\u003eCSH\u003c/em\u003e genes. On the other hand, substitutions are observed in the proximal Pit-1 binding site in the \u003cem\u003eGH-V\u003c/em\u003e genes. Likewise, the TRE shows considerable variation among the genes. On the other hand, the CRE is well conserved, having only two substitutions in the orangutan \u003cem\u003eGH-N\u003c/em\u003e gene, one in the gorilla \u003cem\u003eCSH-B\u003c/em\u003e gene, and one in each of the orangutan \u003cem\u003eGH-V, CSH-L\u003c/em\u003e, and \u003cem\u003eCSH-B\u003c/em\u003e genes. The InrE shows substitutions mainly in the \u003cem\u003eCSH\u003c/em\u003e and the \u003cem\u003eGH-V\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003eThe placental enhancers are located downstream from the \u003cem\u003eCSH\u003c/em\u003e genes. They are well conserved and display all the typical organization in four domains (DF-1 to DF-4). only a few substitutions are observed in its DF-1 and DF-2 domains. A substitution is present in DF-1 (position 16) in the \u003cem\u003eCSH-B\u003c/em\u003e genes\u0026rsquo; enhancers (except for the orangutan). These enhancers also show deletions at the upstream region of the DF-2 domain in man, neanderthal, chimpanzee, and gorilla. Moreover, a substitution in position 65 is also present in these enhancers. With respect to the domains DF-3 and DF-4, they show a few mutations, particularly a substitution in DF-3 in the position 228. Other mutations are also present in the primate \u003cem\u003eCSH\u003c/em\u003e enhancer sequences, like the ones observed at positions 112 and 134. However, these mutations are not inside these enhancer\u0026rsquo;s domains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene content and diversity of encoded proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genes on the apes\u0026rsquo; \u003cem\u003eGH loci\u003c/em\u003e code for three distinctive types of proteins: the pituitary GH-N, the placental GH-V, and CSHs. The \u003cem\u003eGH-N\u003c/em\u003e and \u003cem\u003eGH-V\u003c/em\u003e genes are constants, code for well differentiated proteins, and there is only one copy of each in each \u003cem\u003elocus\u003c/em\u003e. The protein encoded by the \u003cem\u003eGH-N\u003c/em\u003e gene is the most well conserved of all proteins coded by the \u003cem\u003eloci\u003c/em\u003e of all the apes, being identical in human, neanderthal, chimpanzee, gorilla, and orangutan, although surprisingly it has 4\u0026ndash;5 changes in gibbon (see blue boxes in table 3). The protein encoded by the \u003cem\u003eGH-V\u003c/em\u003e gene is also well conserved showing 0, 1, 4, 11 and 14 amino acid changes in neanderthal, chimpanzee, gorilla, orangutan, and gibbon, respectively (see pink boxes in table 3). In contrast to the \u003cem\u003eGH\u003c/em\u003e genes, the \u003cem\u003eCSH\u003c/em\u003e genes differ in number and in their capacity to render protein diversity, since the encoded proteins in a few of these genes are in some cases identical (such as in the case of the two functional genes of the human \u003cem\u003elocus\u003c/em\u003e), and in other apes the gene following the \u003cem\u003eGH-N\u003c/em\u003e, the so called \u003cem\u003eCSH-L\u003c/em\u003e, no longer contributes protein since it became a pseudogene. Also, from the point of view of diversity, the proteins encoded by the \u003cem\u003eCSH\u003c/em\u003e genes are more diverse, as it is shown in the orange boxes in table 3. While \u003cem\u003eCSH\u003c/em\u003e genes in number vary from one to four, the number of different encoded proteins does not follow the gene count. For example, in the human there are \u003cem\u003eCSH\u003c/em\u003e genes: \u003cem\u003eCSH-L\u003c/em\u003e, CSH-A, and \u003cem\u003eCSH-B\u003c/em\u003e, but with the first being a pseudogene and the two-remaining encoding the same mature protein (see red box in table 3), the gene count of three gets reduced to a protein count of one. Thus, five \u003cem\u003eGH-CSH\u003c/em\u003e human genes produces three proteins (one GH-N, one GH-V, and one CSH). A similar pattern is seen in the neanderthal. Since in the chimpanzee, the \u003cem\u003eCSH-A1\u003c/em\u003e gene produces the same mature protein that does the \u003cem\u003eCSH-A2\u003c/em\u003e (red box in table 3), its \u003cem\u003eGH locus\u003c/em\u003e renders four proteins: one GH-N, one GH-V, and two different CSHs. Then, for the gorilla \u003cem\u003eCSH\u003c/em\u003e genes, three different mature proteins derive, since its \u003cem\u003eCSH-A2\u003c/em\u003e gene produces the same protein than its \u003cem\u003eCSH-A3\u003c/em\u003e gene (red box in table 3). Thus, the gorilla \u003cem\u003eGH locus\u003c/em\u003e produces five proteins: one GH-N, one GH-V, and three CSHs. Orangutan \u003cem\u003eGH locus\u003c/em\u003e has two \u003cem\u003eCSH\u003c/em\u003e genes, but one is a pseudogene. Thus, the orangutan \u003cem\u003eGH locus\u003c/em\u003e produces three proteins: one GH-N, one GH-V, and one CSH. Finally, the gibbon has four \u003cem\u003eCSH\u003c/em\u003e genes being one of them also a pseudogene and the rest do not show obvious mutations that could suggest failure in their expression. Therefore, the gibbon \u003cem\u003eGH locus\u003c/em\u003e apparently produces six proteins: two GH-N, one GH-V, and three CSHs. A summary of these gene counts versus potentiality to render diverse proteins is depicted in table 3.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding the evolutionary forces that shaped the multigene family of GH and PRL in mammals have been a subject of great interest. In this study, we have investigated the gene composition and arrangement of anthropoidean \u003cem\u003eGH loci\u003c/em\u003e. To explain their present-day anatomy, we have invoked gene duplications and specializations events, which could have giving rise to the \u003cem\u003eGH\u003c/em\u003e family in higher primates. Our phylogenetic analysis approach is broader, covering the genomic architecture of the \u003cem\u003eloci\u003c/em\u003e in these species, the inspection of the synonymous (dS) versus nonsynonymous (dN) substitution ratios in their nucleotide sequences [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and the conceptual translation of their genes to unravel their coding diversity potential.\u003c/p\u003e \u003cp\u003eThe anatomy of the \u003cem\u003eGH loci\u003c/em\u003e in primates as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that the organization of their gene members in the following general order: \u003cem\u003eGH-N\u003c/em\u003e, \u003cem\u003eCSH-A\u003c/em\u003e (variable number), \u003cem\u003eGH-V\u003c/em\u003e, and \u003cem\u003eCSH-B\u003c/em\u003e. The \u003cem\u003eloci\u003c/em\u003e always begin with \u003cem\u003eGH-N\u003c/em\u003e, then follows \u003cem\u003eCSHs\u003c/em\u003e (including the \u003cem\u003eCSH-L)\u003c/em\u003e, \u003cem\u003eGH-V\u003c/em\u003e, and finally one more \u003cem\u003eCSH\u003c/em\u003e gene. All these \u003cem\u003eloci\u003c/em\u003e, including all known non-primate \u003cem\u003eloci\u003c/em\u003e, are flanked by the \u003cem\u003eCD79B\u003c/em\u003e gene at the 5\u0026rsquo; end by the \u003cem\u003eICAM-1\u003c/em\u003e gene at the 3\u0026rsquo; end. The \u003cem\u003eGH loci\u003c/em\u003e of the analyzed species of apes have several repetitive sequences of the \u003cem\u003eAlu\u003c/em\u003e family in an average of one every 1,000 pb. It is well known that this type of repetitive sequences favors the non-homologous recombination events that originate multigene families. Given that in primates the flanking genes do not change much, \u003cem\u003eGH-N\u003c/em\u003e always is the first gene, and the presence of numerous \u003cem\u003eAlu\u003c/em\u003e sequences, it can be inferred that duplications events and nucleotide changes that originated this family occurred only within the \u003cem\u003eGH\u003c/em\u003e loci. The way in how it happened might well resulted from the alternative evolutionary routes proposed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phylogenetic analysis shown in the tree of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays four well defined and well supported clades by the bootstrap analysis, being from top to bottom \u003cem\u003eGH-N\u003c/em\u003e, \u003cem\u003eGH-V\u003c/em\u003e, \u003cem\u003eCSH\u003c/em\u003e, and non-primate \u003cem\u003eGH\u003c/em\u003e (out-group). It indicates that \u003cem\u003eGH-N\u003c/em\u003e is the orthologous gene, while \u003cem\u003eGH-V\u003c/em\u003e and the \u003cem\u003eCSHs\u003c/em\u003e are the paralogous ones. This analysis supports the evolutionary pathway depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, with two possible routes. In the first route, the ancestral \u003cem\u003eGH-N\u003c/em\u003e gene duplicated to give rise to \u003cem\u003eGH-N\u003c/em\u003e like genes. Then, these experienced a series of mutations that gave rise to the placental genes (\u003cem\u003eGH-V\u003c/em\u003e and \u003cem\u003eCSHs\u003c/em\u003e), and later to the pseudogenization of a \u003cem\u003eCSH\u003c/em\u003e gene. In a variation of this route, the ancestral \u003cem\u003eGH-N\u003c/em\u003e duplicated in one pre-\u003cem\u003eGH-V\u003c/em\u003e gene and some pre-\u003cem\u003eCSH\u003c/em\u003e genes; then a second round of duplication only acting at the pre-\u003cem\u003eCSH\u003c/em\u003e genes gave rise to the various of \u003cem\u003eCSH\u003c/em\u003e genes. Likewise, further mutations in the pre-\u003cem\u003eGH-V\u003c/em\u003e resulted in the \u003cem\u003eGH-V\u003c/em\u003e. Finally, a splicing mutation converted the \u003cem\u003eCSH-L\u003c/em\u003e gene in a pseudogene.\u003c/p\u003e \u003cp\u003eAnother striking feature of the analyzed \u003cem\u003eloci\u003c/em\u003e is the diversity of their encoded hormones. We found that \u003cem\u003eCSH\u003c/em\u003e genes quantity does not match with the CSH mature protein diversity that they encode. Human and neanderthal have three \u003cem\u003eCSH\u003c/em\u003e genes and they encode only one CSH mature protein. Chimpanzee has three \u003cem\u003eCSH\u003c/em\u003e genes and they encode two CSH mature proteins. Gorilla has four \u003cem\u003eCSH\u003c/em\u003e genes and they encode three CSH mature proteins. Orangutan has two \u003cem\u003eCSH\u003c/em\u003e genes and they encode one CSH mature protein. Finally, the gibbon has four \u003cem\u003eCSH\u003c/em\u003e genes and they encode three CSH mature proteins (table 3). This fact opens two interesting questions, such as: 1) why two genes encode the same protein? And 2) do they produce the same protein quantities? First question could be answered by the phylogenetic analysis performed previously [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. They indicate that the evolutionary forces that underling the \u003cem\u003eGH locus\u003c/em\u003e in higher primates is the purifying selection (dN\u0026thinsp;\u0026lt;\u0026thinsp;dS), this force being typical of functional genes. Although we lack information to solve the second question in all apes, we explored it in human and found that they produce similar amounts [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Although this could be an answer to the need to produce surprisingly high amounts of their encoded identical proteins, reaching a couple of grams at the end of pregnancy [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] to fully answer these and other related questions, expression profiles in the apes\u0026rsquo; hypophysis and placenta might be needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to express their gratitude to Ana Karen S\u0026aacute;nchez and Abigail Torres Cerda for their valuable technical assistance with bioinformatic analyses. They also acknowledge the support of the Mexico\u0026rsquo;s National System of Researchers (SNI) and of their respective academic institutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuising, M. O., C. P. Kruiswijk and G. Flik, (2006) Phylogeny and evolution of class-I helical cytokines. J Endocrinol 189: 1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-S\u0026aacute;nchez, J., J. A. Calduch-Giner, M. Mingarro, S. Vega-Rub\u0026iacute;n de Celis, P. G\u0026oacute;mez-Requeni \u003cem\u003eet al.\u003c/em\u003e, (2002) Overview of Fish Growth Hormone Family. New Insights in Genomic Organization and Heterogeneity of Growth Hormone Receptors. Fish Physiology and Biochemistry 27: 243\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePower, D. M., (2005) Developmental ontogeny of prolactin and its receptor in fish. Gen Comp Endocrinol 142: 25\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhittington, C. M., and A. B. Wilson, (2013) The role of prolactin in fish reproduction. Gen Comp Endocrinol 191: 123\u0026ndash;136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBole-Feysot, C., V. Goffin, M. Edery, N. Binart and P. A. Kelly, (1998) Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19: 225\u0026ndash;268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForsyth, I. A., and M. Wallis, (2002) Growth hormone and prolactin\u0026ndash;molecular and functional evolution. J Mammary Gland Biol Neoplasia 7: 291\u0026ndash;312.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawauchi, H., and S. A. Sower, (2006) The dawn and evolution of hormones in the adenohypophysis. Gen Comp Endocrinol 148: 3\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, E. Y., Y. C. Liao, D. H. Smith, H. A. Barrera-Saldana, R. E. Gelinas \u003cem\u003eet al.\u003c/em\u003e, (1989) The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 4: 479\u0026ndash;497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallis, O. C., and M. Wallis, (2002) Characterisation of the GH gene cluster in a new-world monkey, the marmoset (Callithrix jacchus). J Mol Endocrinol 29: 89\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRevol De Mendoza, A., D. Esquivel Escobedo, I. Martinez Davila and H. Saldana, (2004) Expansion and divergence of the GH locus between spider monkey and chimpanzee. Gene 336: 185\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallis, O. C., Y. P. Zhang and M. Wallis, (2001) Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change. J Mol Endocrinol 26: 249\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallis, O. C., A. O. Mac-Kwashie, G. Makri and M. Wallis, (2005) Molecular evolution of prolactin in primates. J Mol Evol 60: 606\u0026ndash;614.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWallis, O. C., and M. Wallis, (2006) Evolution of growth hormone in primates: the GH gene clusters of the New World monkeys marmoset (Callithrix jacchus) and white-fronted capuchin (Cebus albifrons). J Mol Evol 63: 591\u0026ndash;601.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapper, Z., N. M. Jameson, R. Romero, A. L. Weckle, P. Mittal \u003cem\u003eet al.\u003c/em\u003e, (2009) Ancient origin of placental expression in the growth hormone genes of anthropoid primates. Proc Natl Acad Sci U S A 106: 17083\u0026ndash;17088.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGluckman, P. D., and C. S. Pinal, (2002) Maternal-placental-fetal interactions in the endocrine regulation of fetal growth: role of somatotrophic axes. Endocrine 19: 81\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDattani, M., and M. Preece, (2004) Growth hormone deficiency and related disorders: insights into causation, diagnosis, and treatment. Lancet 363: 1977\u0026ndash;1987.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, Y., B. C. Xu, H. G. Maheshwari, L. He, M. Reed \u003cem\u003eet al.\u003c/em\u003e, (1997) A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci U S A 94: 13215\u0026ndash;13220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliot, M. G., and B. J. Crespi, (2008) Placental invasiveness and brain-body allometry in eutherian mammals. J Evol Biol 21: 1763\u0026ndash;1778.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaig, D., (2008) Placental growth hormone-related proteins and prolactin-related proteins. Placenta 29 Suppl A: S36-41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez Alvarez, R., A. Revol de Mendoza, D. Esquivel Escobedo, G. Corrales Felix, I. Rodriguez Sanchez \u003cem\u003eet al.\u003c/em\u003e, (2006) Growth hormone locus expands and diverges after the separation of New and Old World Monkeys. Gene 380: 38\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLacroix, M. C., J. Guibourdenche, T. Fournier, I. Laurendeau, A. Igout \u003cem\u003eet al.\u003c/em\u003e, (2005) Stimulation of human trophoblast invasion by placental growth hormone. Endocrinology 146: 2434\u0026ndash;2444.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFleenor, D., J. Oden, P. A. Kelly, S. Mohan, S. Alliouachene \u003cem\u003eet al.\u003c/em\u003e, (2005) Roles of the lactogens and somatogens in perinatal and postnatal metabolism and growth: studies of a novel mouse model combining lactogen resistance and growth hormone deficiency. Endocrinology 146: 103\u0026ndash;112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Ibave, D. C., I. P. Rodr\u0026iacute;guez-S\u0026aacute;nchez, L. Garza-Rodr\u0026iacute;guez Mde and H. A. Barrera-Salda\u0026ntilde;a, (2014) Extrapituitary growth hormone synthesis in humans. Growth Horm IGF Res 24: 47\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Ibave, D. C., I. P. Rodr\u0026iacute;guez-S\u0026aacute;nchez, M. L. Garza-Rodr\u0026iacute;guez, A. A. P\u0026eacute;rez-Maya, M. Luna \u003cem\u003eet al.\u003c/em\u003e, (2018) Expression of growth hormone gene in the baboon eye. Exp Eye Res 169: 157\u0026ndash;169.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Maya, A. A., I. P. Rodr\u0026iacute;guez-S\u0026aacute;nchez, P. de Jong, M. Wallis and H. A. Barrera-Salda\u0026ntilde;a, (2012) The chimpanzee GH locus: composition, organization, and evolution. Mamm Genome 23: 387\u0026ndash;398.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Maya, A. A., M. Wallis and H. A. Barrera-Salda\u0026ntilde;a, (2016) Structure and evolution of the gorilla and orangutan growth hormone loci. Mamm Genome 27: 511\u0026ndash;523.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmit, A., Hubley, R \u0026amp; Green P, (2013\u0026ndash;2015) RepeatMasker Open-4.0, pp.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChenna, R., H. Sugawara, T. Koike, R. Lopez, T. J. Gibson \u003cem\u003eet al.\u003c/em\u003e, (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31: 3497\u0026ndash;3500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar, (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 30: 2725\u0026ndash;2729.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaitou, N., and M. Nei, (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406\u0026ndash;425.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez-Rodriguez, H. G., Guerra-Rodriguez, N. E., Iturbe-Cantu, M. A., Martinez-Torres, A., \u0026amp; Barrera-Salda\u0026ntilde;a, H. A. (1997). Expression of human placental lactogen and variant growth hormone genes in placentas. Arch Med Res, 28(4), 507\u0026ndash;512.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrera-Salda\u0026ntilde;a HA, Seeburg PH, Saunders GF (1983) Two structurally different genes produce the same secreted human placental lactogen hormone. The Journal of biological chemistry, 258(6):3787\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"table","content":"\u003cp\u003eTable 3 is available in the Supplementary Files section.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chorionic somatomammotropins, somatolactins, prolactin, platyrrhine, cercopithecidae, monkeys","lastPublishedDoi":"10.21203/rs.3.rs-1844540/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1844540/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrowth hormone (GH) \u003cem\u003elocus\u003c/em\u003e experienced dramatic evolution in primates, becoming multigenic and diverse in anthropoids. Despite sequence information from vast number of primate species, it has remained unclear how the multigene family was favored feature. We sought to compare the structure and composition of the apes\u0026rsquo; \u003cem\u003eGH loci\u003c/em\u003e as a prerequisite to understanding their origin and possible evolutionary role. \u003cem\u003eGH loci\u003c/em\u003e of chimpanzee, gorilla, and orangutan were sequenced from bacterial artificial chromosomes, while those of modern man, neanderthal, gibbon, and wild boar were retrieved from GenBank. Coding regions, regulatory elements, and repetitive sequences were identified and compared among species. GH \u003cem\u003eloci\u003c/em\u003e of all analyzed species are flanked by the genes \u003cem\u003eCD79B\u003c/em\u003e (5\u0026acute;) and \u003cem\u003eICAM-1\u003c/em\u003e(3\u0026acute;). In man, neanderthal, and chimpanzee the \u003cem\u003eloci\u003c/em\u003e are integrated by five genes almost indistinguishable, however while in the former two they render three different hormones, in the latter one four different proteins are derived. Gorilla exhibits six genes, gibbon seven, and orangutan four. The sequences of proximal promoters, enhancers, P-elements, and a locus control region (LCR) are highly conserved. The locus evolution might have implicated duplications of the ancestral pituitary \u003cem\u003eGH-N\u003c/em\u003e gene and subsequent diversification of the copies, to lead to the placental single \u003cem\u003eGH-V\u003c/em\u003e gene and to the multiple also placental \u003cem\u003eCSH\u003c/em\u003e genes.\u003c/p\u003e","manuscriptTitle":"Gene content and coding diversity of growth hormone loci of apes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-19 16:29:54","doi":"10.21203/rs.3.rs-1844540/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3f9d771-3f9c-4e75-9ddb-bc5b323e184f","owner":[],"postedDate":"July 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-10-04T15:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-19 16:29:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1844540","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1844540","identity":"rs-1844540","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}