Vertical clinging and leaping revisited: vertical support use as the ancestral condition of strepsirrhine primates

A re-examination of primate foot and knee anatomy suggests that strepsirrhine primates (adapiforms and lemuriforms) possess a unique and derived hindlimb related to their use of vertical supports. In contrast, leaping adaptations are older and shared by both major euprimate clades, Strepsirrhini and Haplorhini. Combining this derived hindlimb anatomy with leaping suggests that ancestral strepsirrhines were at least frequent vertical support users and leapers, and perhaps vertical clingers and leapers. These initial strepsirrhine adaptations were preadaptive for later lemuriform vertical clingers and leapers. In contrast, haplorhine vertical clingers and leapers require additional foot and leg modifications to accommodate a vertical clinging and leaping lifestyle. The movement pattern called vertical clinging and leaping evolved independently among different primate lineages throughout primate evolutionary history for several different ecological reasons.     

Canine relaxin-like factor: unique molecular structure and differential expression within reproductive tissues of the dog

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.     

Molecular cytogenetic studies in strepsirrhine primates, dermoptera and scandentia

Since the first chromosome painting study between human and strepsirrhine primates was performed in 1996, nearly 30 species in Strepsirrhini, Dermoptera and Scandentia have been analyzed by cross-species chromosome painting. Here, the contribution of chromosome painting data to our understanding of primate genome organization, chromosome evolution and the karyotype phylogenetic relationships within strepsirrhine primates, Dermoptera and Scandentia is reviewed. Twenty-six to 43 homologous chromosome segments have been revealed in different species with human chromosome-specific paint probes. Various landmark rearrangements characteristic for each different lineage have been identified, as cytogenetic signatures that potentially unite certain lineages within strepsirrhine primates, Dermoptera and Scandentia.     

B-chain sequence and in situ hybridization of the rabbit placental relaxin-like gene product.

We reported that the nucleotide sequence of a cDNA generated from rabbit placental poly(A)(+) RNA using porcine preprorelaxin primers was identical to SQ10, a product of squamous differentiated tracheal epithelial cells. However, these results did not confirm that SQ10 was the biologically active rabbit relaxin that had been isolated previously yet not sequenced. In this study, a 7-kDa protein isolated from rabbit placentas exhibited relaxin bioactivity and cross-reacted with a porcine relaxin antiserum. A partial amino acid sequence of this protein revealed a sequence identical to that of SQ10. Although the amino acid sequence of the putative relaxin receptor-binding domain found in the B chain of relaxin was modified in SQ10 from CGRDYVR to CRNDFVR, the placental protein was bioactive. These results suggest that SQ10 is the rabbit relaxin. In situ hybridization, using an SQ10 riboprobe, indicated radiolabeling in the syncytiotrophoblast cells of the rabbit placenta. The pattern of labeling corresponded with the immunohistochemical staining for relaxin observed with use of a porcine relaxin antiserum. These results indicate that the syncytiotrophoblast cells are a site of synthesis for SQ10 and that the immunostaining is not solely the result of sequestering SQ10 through receptor-mediated endocytosis. A potential role for relaxin in implantation is discussed.     

Estimation of the Transition/Transversion Rate Bias and Species Sampling

The transition/transversion (ti/tv) rate ratios are estimated by pairwise sequence comparison and joint likelihood analysis using mitochondrial cytochrome b genes of 28 primate species, representing both the Strepsirrhini (lemurs and lories) and the Anthropoidea (monkeys, apes, and humans). Pairwise comparison reveals a strong negative correlation between estimates of the ti/tv ratio and the sequence distance, even when both are corrected for multiple substitutions. The maximum-likelihood estimate of the ti/tv ratio changes with the species included in the analysis. The ti/tv bias within the lemuriform taxa is found to be as strong as in the anthropoids, in contradiction to an earlier study which sampled only one lemuriform. Simulations show the surprising result that both the pairwise correction method and the joint likelihood analysis tend to overcorrect for multiple substitutions and overestimate the ti/tv ratio, especially at low sequence divergence. The bias, however, is not large enough to account for the observed patterns. Nucleotide frequency biases, variation of substitution rates among sites, and different evolutionary dynamics at the three codon positions can be ruled out as possible causes. The likelihood-ratio test suggests that the ti/tv rate ratios may be variable among evolutionary lineages. Without any biological evidence for such a variation, however, we are left with no plausible explanations for the observed patterns other than a possible saturation effect due to the unrealistic nature of the model assumed. Received: 1 October 1997 / Accepted: 29 September 1998     

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.     

The primary structure and the disulfide links of the bovine relaxin-like factor (RLF).

The relaxin-like factor (RLF), produced by the Leydig cells, is an essential link in the chain of events leading to the proper positioning of the testes during fetal development. The primary structure of RLF, as reported in the literature, is based solely upon cDNA sequences with chain lengths determined according to deduced processing sites and with relaxin-like cross-links. Biochemical characterization of bovine testicular RLF shows clearly that the endogenous hormone does consist of a 26 residue A chain and two forms of B chain, one containing 40 residues, the other 45. In addition, both B chains are 9 residues longer at the C terminus than the cDNA-deduced chain, and about 20% of the B chains have an additional 5 residue extension at the N terminus. Sequence analysis in combination with mass spectrometry and tryptic peptide mapping showed unambiguously that RLF is larger than previously assumed and that it has the relaxin-type disulfide bond distribution that makes it a bona fide member of the relaxin family of hormones.     

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.     

The mode of interaction of the relaxin-like factor (RLF) with the leucine-rich repeat G protein-activated receptor 8

The relaxin-like factor (RLF, also named INSL3) is a critical component in the chain of events that lead to the normal positioning of the gonads in the male fetus. RLF and relaxin share features of the secondary structure to the extent that relaxin cross-reacts with the LGR8, the RLF receptor. Although both hormones interact with their receptors essentially via the B chain, the sharply defined binding cassette of relaxin is not present in RLF. Structure and function analysis of RLF derivatives with single amino acid replacements revealed that the most important binding residues are tryptophan B27, followed by arginine B16 and valine B19. Single alanine replacements for each individual position resulted in a relative receptor affinity of 4.0% (B16), 6.1% (B19), and 0.5% (B27). Tryptophan B27 is located on an extended structure, and arginine B16 and valine B19 are positioned on the exposed surface of the B chain helix. The 3 residues could be brought together to form a contiguous binding area if the C-terminal end of the B chain were free to fold back against the central portion of the B chain helix. Such a movement depends critically on the flexibility of the C-terminal end, which is controlled by positions B23-25. In as much as these major binding residues seem hardly sufficient to explain the strong binding of RLF to LGR8 we searched for and found an extended region where little contributions by individual residues added up to a strong receptor affinity. This mode of interaction could drive the binding energy sufficiently high to account for the picomolar binding constant of RLF and its receptor.     

Characterization and Phylogenetic Relationship of Prosimian MHC Class I Genes

MHC class I cDNA sequences from the most divergent primate group of extant primates compared to human, the suborder Strepsirrhini (prosimians), are described. The sequences are derived from the gray mouse lemur (Microcebus murinus) and the ring-tailed lemur (Lemur catta), which are members of the malagasy Lemuriformes, as well as from the pygmy slow loris (Nycticebus pygmaeus), a prosimian from East Asia. The M. murinus sequences have been analyzed in detail. Analysis of the expression level, G/C content, and synonymous vs. nonsynonymous substitution rates in the peptide-binding region codons suggests that these cDNA clones represent classical class I (class Ia) genes. According to Southern blot analysis, the genome of the gray mouse lemur might contain about 10 class I genes. In gene tree analysis, the strepsirrhine class Ia genes described here cluster significantly separately from the known class I genes of Catarrhini (humans, apes, Old World monkeys) and Platyrrhini (New World monkeys) species, suggesting that the class I loci of Simiiformes arose by gene duplications which occurred after the divergence of prosimians.     

Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding

The relaxin-like factor (RLF) is a circulating hormone that is synthesized in the gonads of mammals and released into the bloodstream. The distribution of its receptor and a trace of cross-reactivity to relaxin receptors implied that this relatively new factor is more relaxin- than insulin-like. The chemical synthesis of RLF analogues with specific modifications in positions B27 and B25, or the truncated form des(B27-31)RLF, clearly indicate that the intact indole ring in position B27 is crucial for high RLF receptor-binding. Receptor-binding was reduced by 2 orders of magnitude for Leu(B27)RLF (3%), Ala(B27)RLF (2.1%), and des(B27-31)RLF (0.4%), whereas slightly better binding was observed for His(B27)RLF (7.5%), Phe(B27)RLF (21%), D-Trp(B27) (26%), and the oxindole(B27)RLF (41%). On the basis of these observation it seems that an aromatic ring system in the beta- or gamma-position is required for binding. Structure prediction of the C-terminal region of the B chain indicated a possible type I or type III turn for residues C-G-G-P-R (B22-26) preceding the tryptophan. Exchanging Pro(B25) for D-Pro within the turn caused a severe structural rearrangement at the C terminus and a 96% drop in activity. It appears that the steric effect of L-Pro(B25) is important for the proper positioning of Trp(B27).     

Molar scaling in strepsirrhine primates

We examined how maxillary molar dimensions change with body and skull size estimates among 54 species of living and subfossil strepsirrhine primates. Strepsirrhine maxillary molar areas tend to scale with negative allometry, or possibly isometry, relative to body mass. This observation supports several previous scaling analyses showing that primate molar areas scale at or slightly below geometric similarity relative to body mass. Strepsirrhine molar areas do not change relative to body mass(0.75), as predicted by the metabolic scaling hypothesis. Relative to basicranial length, maxillary molar areas tend to scale with positive allometry. Previous claims that primate molar areas scale with positive allometry relative to body mass appear to rest on the incorrect assumption that skull dimensions scale isometrically with body mass. We identified specific factors that help us to better understand these observed scaling patterns. Lorisiform and lemuriform maxillary molar scaling patterns did not differ significantly, suggesting that the two infraorders had little independent influence on strepsirrhine scaling patterns. Contrary to many previous studies of primate dental allometry, we found little evidence for significant differences in molar area scaling patterns among frugivorous, folivorous, and insectivorous groups. We were able to distinguish folivorous species from frugivorous and insectivorous taxa by comparing M1 lengths and widths. Folivores tend to have a mesiodistally elongated M1 for a given buccolingual M1 width when compared to the other two dietary groups. It has recently been shown that brain mass has a strong influence on primate dental eruption rates. We extended this comparison to relative maxillary molar sizes, but found that brain mass appears to have little influence on the size of strepsirrhine molars. Alternatively, we observed a strong correlation between the relative size of the facial skull and relative molar areas among strepsirrhines. We hypothesize that this association may be underlain by a partial sharing of the patterning of development between molar and facial skull elements.     

Linguistic measure of taxonomic and functional relatedness of nucleotide sequences

The frequencies of "words", oligonucleotides within nucleotide sequences, reflect the genetic information contained in the sequence "texts". Nucleotide sequences are characteristically represented by their contrast word vocabularies. Comparison of the sequences by correlating their contrast vocabularies is shown to reflect well the relatedness (unrelatedness) between the sequences. A single value, the linguistic similarity between the sequences, is suggested as a measure of sequence relatedness. Sequences as short as 1000 bases can be characterized and quantitatively related to other sequences by this technique. The linguistic sequence similarity value is used for analysis of taxonomically and functionally diverse nucleotide sequences. The similarity value is shown to be very sensitive to the relatedness of the source species, thus providing a convenient tool for taxonomic classification of species by their sequence vocabularies. Functionally diverse sequences appear distinct by their linguistic similarity values. This can be a basis for a quick screening technique for functional characterization of the sequences and for mapping functionally distinct regions in long sequences.     

Patterns of astragalar fibular facet orientation in extant and fossil primates and their evolutionary implications

A laterally sloping fibular facet of the astragalus (=talus) has been proposed as one of few osteological synapomorphies of strepsirrhine primates, but the feature has never been comprehensively quantified. We describe a method for calculating fibular facet orientation on digital models of astragali as the angle between the planes of the fibular facet and the lateral tibial facet. We calculated this value in a sample that includes all major extant primate clades, a diversity of Paleogene primates, and nonprimate euarchontans (n = 304). Results show that previous characterization of a divide between extant haplorhines and strepsirrhines is accurate, with little overlap even when individual data points are considered. Fibular facet orientation is conserved in extant strepsirrhines despite major differences in locomotion and body size, while extant anthropoids are more variable (e.g., low values for catarrhines relative to non‐callitrichine platyrrhines). Euprimate outgroups exhibit a mosaic of character states with Cynocephalus having a more obtuse strepsirrhine‐like facet and sampled treeshrews and plesiadapiforms having more acute haplorhine‐like facets. Surprisingly, the earliest species of the adapiform Cantius have steep haplorhine‐like facets as well. We used a Bayesian approach to reconstruct the evolution of fibular facet orientation as a continuous character across a supertree of living and extinct primates. Mean estimates for crown Primatomorpha (97.9°), Primates (99.5°), Haplorhini (98.7°), and Strepsirrhini (108.2°) support the hypothesis that the strepsirrhine condition is derived, while lower values for crown Anthropoidea (92.8°) and Catarrhini (88.9°) are derived in the opposite direction. Am J Phys Anthropol 151:420–447, 2013.© 2013 Wiley Periodicals, Inc.     

Identification of the blood group Lewis(a) determinant in the oviducal mucins of Xenopus tropicalis

The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewis(a) epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of (1)H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis(a)-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(beta 1-3)GlcNAc(beta 1-6)GalNAc-ol. These structural data suggested the emergence of an alpha 1,4-fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis-fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.