Comprehensive analysis and characterization of the TCR α chain sequences in the common marmoset

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR α constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68–100%, and 79–98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRβ chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d _S ) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d _N ) were significantly lower in the FRs than in CDRs. Interestingly, the d _N values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRβ than in CDRα by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.     

Development and annotation of shotgun sequence libraries from New World monkeys

The draft genome sequences of several primates are available, providing insights into evolutionary and anthropological research. However, genomic resources from New World monkeys are conspicuously lacking. To date, the genomes of only two platyrrhine species, the common marmoset and the Bolivian squirrel monkey, have been fully sequenced. This is especially limiting for comparative genomics research, considering that New World monkeys are the most speciose primate group, and platyrrhine genetic diversity is comparable to that of the catarrhines (i.e. apes and Old World monkeys). Here, we present the generation and annotation of numerous sequence reads from the genomes of Spider monkey (Ateles belzebuth), Owl monkey (Aotus lemurinus) and Uakari (Cacajao calvus), representing the three platyrrhine families, Atelidae, Cebidae and Pitheciidae, respectively. These sequencing reads were developed from gDNA shotgun libraries containing over 3000 individual sequences with an average length of 726 bps. Of these sequences, 1220 contain <20% repeats, and thus are potentially highly useful phylogenetic markers for other platyrrhine species. Among them, a large number of sequencing reads were found to match unique regions within the human (2462 sequences) and the marmoset (2829 sequences) genomes. In particular, the majority of these sequencing reads are from putatively neutrally evolving intergenic regions. Thus, they are likely to be highly informative for inferring neutral evolutionary patterns and genomic evolution for other New World monkeys.     

ASPM and the Evolution of Cerebral Cortical Size in a Community of New World Monkeys

The ASPM (abnormal spindle-like microcephaly associated) gene has been proposed as a major determinant of cerebral cortical size among primates, including humans. Yet the specific functions of ASPM and its connection to human intelligence remain controversial. This debate is limited in part by a taxonomic focus on Old World monkeys and apes. Here we expand the comparative context of ASPM sequence analyses with a study of New World monkeys, a radiation of primates in which enlarged brain size has evolved in parallel in spider monkeys (genus Ateles) and capuchins (genus Cebus). The primate community of Costa Rica is perhaps a model system because it allows for independent pairwise comparisons of smaller- and larger-brained species within two taxonomic families. Accordingly, we analyzed the complete sequence of exon 18 of ASPM in Ateles geoffroyi, Alouatta palliata, Cebus capucinus, and Saimiri oerstedii. As the analysis of multiple species in a genus improves phylogenetic reconstruction, we also analyzed eleven published sequences from other New World monkeys. Our exon-wide, lineage-specific analysis of eleven genera and the ratio of rates of nonsynonymous to synonymous substitutions (d_N/d_S) on ASPM revealed no detectable evidence for positive selection in the lineages leading to Ateles or Cebus, as indicated by d_N/d_S ratios of <1.0 (0.6502 and 0.4268, respectively). Our results suggest that a multitude of interacting genes have driven the evolution of larger brains among primates, with different genes involved in this process in different encephalized lineages, or at least with evidence for positive selection not readily apparent for the same genes in all lineages. The primate community of Costa Rica may serve as a model system for future studies that aim to elucidate the molecular mechanisms underlying cognitive capacity and cortical size.     

Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae) and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in d_N/d_S in Setophaga, implying relatively strong selective pressures to overcome long‐standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus, substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark–light adaptation. Although it is unclear what these alterations mean for colour perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga.     

The T-cell receptor β chain-encoding gene repertoire of a New World primate species,the cotton-top tamarin

 The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species’ unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8⁺ T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR β chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (p_N) and synonymous (p_S) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in p_N in the framework regions compared with p_N in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR β chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts. Received: 19 July 1996 / Revised: 12 August 1996     

Williams-Beuren mapping in Callithrix argentata, Callicebus cupreus and Alouatta caraya indicates different patterns of chromosomal rearrangements in neotropical primates

Human chromosome 7 has a complex syntenic origin. It was divided into two segments in both the ancestral primate karyotype and in Platyrrhini. Apparently, a small segment in the ancestral platyrrhine karyotype was associated with HSA5 and the remainder formed a middle‐sized submetacentric. We tested the dynamics of platyrrhine chromosomes by hybridizing the locus specific Willams‐Beuren probe (7q 11.23, 450 kb) to chromosomes of representative species from the three families of the New World monkeys recently proposed by molecular genomics: Cebidae, Callithrix argentata (bare ear marmoset or silvery marmoset, 2n = 44); Pitheciidae, Callicebus cupreus [red titi monkey, or coppery monkey, 2n = 46)] and Atelidae, Alouatta caraya (black and gold howler, 2n = 52). In both the marmoset and the howler monkeys, the signal was found on the small segment of chromosome 7 associated with human chromosome 5, but not in Callicebus cupreus. Instead, the Williams‐Beuren syndrome (WS) signal was found on a C. cupreus chromosome previously reported to be hybridized only by human chromosome 1. The WS probe indicates a small, but complex translocation never described before. Our results point out that fluorescence in situ hybridization (FISH) with locus specific probes and cloned DNA fragments such as bacterial aftificial chromosomes (BACs) provides higher resolution than FISH with whole chromosomes paints. It may be well that the variability seen in the hybridization patterns and revealed by the WS FISH in this report is as a result of a rearrangement ‘hot spot’. The WS region in humans is composed of region‐specific different blocks of complex segmental duplications that probably promote the extraordinary rate of evolutionary dynamics of this region among primate species, and which continues to be reflected today by the predisposition of this region to disease syndromes such as WS. The evolutionary history of this region also suggests that repeat families in this region had their origin in a common ancestor of both Old World and New World monkeys.     

Inflammatory vulnerability associated with the rh5‐HTTLPR genotype in juvenile rhesus monkeys

Individual variation in serotonergic function is associated with reactivity, risk for affective disorders, as well as an altered response to disease. Our study used a nonhuman primate model to further investigate whether a functional polymorphism in the promoter region for the serotonin transporter gene helps to explain differences in proinflammatory responses. Homology between the human and rhesus monkey polymorphisms provided the opportunity to determine how this genetic variation influences the relationship between a psychosocial stressor and immune responsiveness. Leukocyte numbers in blood and interleukin‐6 (IL‐6) responses are sensitive to stressful challenges and are indicative of immune status. The neutrophil‐to‐lymphocyte ratio and cellular IL‐6 responses to in vitro lipopolysaccharide stimulation were assessed in 27 juvenile male rhesus monkeys while housed in stable social groups (N_LL = 16, N_S = 11) and also in 18 animals after relocation to novel housing (N_LL = 13, N_S = 5). Short allele monkeys had significantly higher neutrophil‐to‐lymphocyte ratios than homozygous Long allele carriers at baseline [t(25) = 2.18, P = 0.02], indicative of an aroused state even in the absence of disturbance. In addition, following the housing manipulation, IL‐6 responses were more inhibited in short allele carriers (F_1,16 = 8.59, P = 0.01). The findings confirm that the serotonin transporter gene‐linked polymorphism is a distinctive marker of reactivity and inflammatory bias, perhaps in a more consistent manner in monkeys than found in many human studies .     

Comparison of marmoset and human FSH using synthetic peptides of the β‐subunit L2 loop region and anti‐peptide antibodies

Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH–FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti‐peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β‐subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32–50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone–receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross‐species peptide and FSH in ELISA. The anti‐peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH–FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Difference in larval type explains patterns of nonsynonymous substitutions in two ancient paralogs of the histone H3 gene in sea stars

SUMMARY Paralogous genes frequently show differences in patterns and rates of substitution that are typically attributed to different selection regimes, mutation rates, or local recombination rates. Here, two anciently diverged paralogous copies of the histone H3 gene in sea stars, the tandem‐repetitive early‐stage gene and a newly isolated gene with lower copy number that was termed the “putative late‐stage histone H3 gene” were analyzed in 69 species with varying mode of larval development. The two genes showed differences in relative copy number, overall substitution rates, nucleotide composition, and codon usage, but similar patterns of relative nonsynonymous substitution rates, when analyzed by the d_N/d_S ratio. Sea stars with a nonpelagic and nonfeeding larval type (i.e., brooding lineages) were observed to have d_N/d_S ratios that were larger than for nonbrooders but equal between the two paralogs. This finding suggested that demographic differences between brooding and nonbrooding lineages were responsible for the elevated d_N/d_S ratios observed for brooders and refuted a suggestion from a previous analysis of the early‐stage gene that the excess nonsynonymous substitutions were due to either (1) gene expression differences at the larval stage between brooders and nonbrooders or (2) the highly repetitive structure of the early‐stage histone H3 gene.     

Identification of New World monkey MHC-DRB alleles using PCR,DGGE and direct sequencing

Identification of New World monkey MHC-DRB alleles has previously relied upon labor-intensive cloning and sequencing techniques. Here we describe a rapid and unambiguous way to distinguish DRB alleles in New World monkeys using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of New World monkey DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. The validity of this typing procedure was confirmed by the identification of all DRB alleles previously characterized by cloning and sequencing techniques from an individual cotton-top tamarin. Importantly, our analysis revealed DRB alleles not previously identified in this reference animal. Following validation of our technique, the protocol was employed for the characterization of MHC-DRB alleles in four other species of New World monkey: the pygmy marmoset, white-faced saki monkey, long-haired spider monkey and owl monkey. Using this technique, we identified five alleles from the cotton-top tamarin, five alleles from the owl monkey, three alleles from the long-haired spider monkey, three alleles from the white-faced saki monkey and two alleles from the pygmy marmoset. On the basis of phylogenetic tree analyses, 13 new DRB alleles were assigned to eight different MHC-DRB lineages. Whereas traditional DRB typing via cloning and sequencing provides limited information, our new technique provides a simple and relatively rapid way of identifying New World monkey MHC-DRB alleles.Nucleotide sequence data reported are available in the GenBank/EMBL/DDBJ databases under the accession numbers AJ544165–AJ544177     

Molecular Evolution of Prolactin in Primates

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.     

The chromosomal location of rDNA in selected lower primates.

Hybridization in stiu was used to identify the chromosomes that carry rDNA in representative lower primates, including the baboons, Papio cynocephalus and Papio hamadryas; the colobus monkey, Colobus polykomos; the tree shrew, Tupaia glis; the lemur, Lemur fulvis; the saki, Pithecia pithecia; the marmoset, Saguinus nigricollis, and the spider monkey, Ateles geoffroyi. The marker chromosome, common to the Cercopithecines studied to date, carries the rDNA in the baboons. Another marker chromosome carries rDNA in a South American species, the spider monkey. A multichromosomal distribution of rDNA was demonstrated in the tree shrew, lemur, saki, and marmoset. None of the rDNA-containing chromosomes in the prosimians and New World monkeys show homology to the chromosomes that carry rDNA in the Hominids, Pongids, or Old World monkeys.     

Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease

Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4⁺-T-cell receptor beta variable (TRBV) 29-1⁺ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4^- (TRAV9-2⁺) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4⁺-TRBV29-1⁺ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2⁺-TRBV7-3⁺ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4⁺ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2⁺ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3β loop of the TCR and Arg70β from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.     

Development of an enzyme-immunoassay (EIA) for the measurement of follicle-stimulating-hormone (FSH) in callitrichid primates using a monoclonal antibody against the human-FSH-β-subunit

Despite the importance of Callitrichid primates in both biomedical and conservation research, practical and reliable immunoassays for the measurement of follicle-stimulating hormone (FSH) have not yet been described. A panel of monoclonal antibodies against specific peptide fragments within either the alpha or beta subunit of human FSH was evaluated for their ability to recognize FSH from Callitrichid and other New World primates. One of these, monoclonal antibody 46.3h6.b7 raised against human FSH, was selected due to its ability to recognize marmoset monkey FSH and its low crossreactivity with other gonadotrophins. The antibody formed the basis of an enzymeimmunoassay using a highly purified human urinary FSH (Metrodin®, Serono) preparation coupled to biotin as label and unmodified as standard. After 24 h incubation, antibody bound label was visualized by addition of streptavidin-peroxidase followed by the appropriate substrate. Parallelism was obtained between the standard and dilutions of pituitary extracts, urine and plasma from the common marmoset (Callithrix jacchus) as well as from two tamarin species (Saguinus fuscicollis and S. oedipus) and one squirrel monkey (Saimiri sciureus). Profiles of plasma and urinary FSH during the follicular phase are shown for two individual marmosets. The ability to measure FSH in Callitrichidae provides new opportunities for studies of the reproductive biology of these New World primate species. Am. J. Primatol. 41:179–193, 1997. © 1997 Wiley-Liss, Inc.     

MHC (Major Histocompatibility Complex)-DRB Genes and Polymorphisms in Common Marmoset

A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes. Received: 18 February 2000 / Accepted: 17 May 2000     

Characterization of T-cell repertoire in hairy cell leukemia patients before and after recombinant immunotoxin BL22 therapy

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.     

Cerebellar localization of the NO-receptive soluble guanylyl cyclase subunits-α2/β1 in non-human primates

Nitric-oxide-sensitive guanylyl cyclase (NO-sGC) plays a pivotal role in many second messenger cascades. Neurotransmission- and neuropathology-related changes in NO-sGC have been suggested. However, the cellular localization of NO-sGC in primate brains, including humans, remains unknown. Biochemical evidence has linked the α₂-subunit of NO-sGC directly to neurotransmission in rodents. Here, we have used a recently characterized subunit-specific antibody for the localization of the α₂-subunit on sections from the cerebelli of the common marmoset (Callithrix jacchus; New World monkey) and macaque monkeys (Macaca mulatta, M. fascicularis; Old World monkeys). In contrast to the more ubiquitous cytoplasmic presence of subunit-β₁, the α₂-subunit is mainly confined to the somato-dendritic membrane including the spines of the Purkinje cells. Only limited colocalization with presynaptically localized synaptophysin has been seen under our staining conditions, indicating a higher abundance of subunit-α₂ at the postsynaptic site. This localization indicates that subunit-α₂ links NO-sGC to neurotransmission, whereas subunit-β₁ may act as a cytoplasmic regulator/activator by contributing to active heterodimer formation via translocation from the cytoplasm to the cell membrane. The last-mentioned action may be a prerequisite for generating nitric-oxide-dependent, subcellular, and postsynaptically localized cGMP signals along neuronal processes.This study was supported by the Deutsche Forschungsgemeinschaft.     

Invertebrate Species with Nonpelagic Larvae Have Elevated Levels of Nonsynonymous Substitutions and Reduced Nucleotide Diversities

Under a nearly neutral model in which most amino acid substitutions are slightly deleterious, variation in demography, population structure, and other ecological factors among closely related species can potentially modify the effective population size or the selective regime, leading to differences in the rate of nonsynonymous substitution. Ratios of nonsynonymous to synonymous substitutions (d_N/d_S) between species were analyzed in a sea star genus (Patiriella) and a molluscan genus (Littorina), each with diverse modes of reproduction, including multiple lineages with pelagic and nonpelagic larvae. In both genera, lineages with nonpelagic larvae had significantly higher d_N/d_S ratios than lineages with pelagic larvae. The hypothesis that the elevated d_N/d_S ratios in species with nonpelagic larvae was due to reduced effective population size was tested by comparing nucleotide diversities in three genera of gastropod mollusks (Littorina, Crepidula, and Hydrobia), each with several modes of reproduction. Overall, there was a significant (p < 0.05) reduction in nucleotide diversity in species with nonpelagic larvae compared to species with pelagic larvae.     

Monomorphic Region of the Serotonin Transporter Promoter Gene in New World Monkeys

Genetic variation in the human serotonin system has long been studied because of its functional consequences and links to various neuropsychiatric and behavior‐related disorders. Among non‐human primates, the common marmosets (Callithrix jacchus) and tufted capuchins monkeys (Cebus apella) are becoming increasingly used as models to study the effects of genes, environments, and their interaction on physiology and complex behavior. In order to investigate the independent functions of and potential interactions between serotonin‐related genes, anxiety and neuropsychiatric disorders, we analyzed the presence and variability of the serotonin transporter gene‐linked polymorphic region (5‐HTTLPR) in marmoset and capuchin monkeys. By PCR and using heterologous primers from the human sequence, we amplified and then sequenced the corresponding 5‐HTT region in marmosets and capuchins. The resulting data revealed the presence of a tandem repeat sequence similar to that described in humans, but unlike humans and other Old World primates, no variable length alleles were detected in these New World monkeys, suggesting that if serotonin transporter is involved in modulating behavior in these animals it does so through different molecular mechanisms. Am. J. Primatol. 74:1028‐1034, 2012. © 2012 Wiley Periodicals, Inc.     

Distribution of human endogenous retrovirus HERV-K genomes in humans and different primates

The distribution of the human endogenous retrovirus (HERV)-K genome was investigated by Southern-blot analyses using a HERV-K-env DNA probe. With the exception of one DNA-sample, obtained from a Chinese individual in whom an amplification of HERV-K was detected, Southern-blot analyses yielded identical hybridization patterns with DNA from peripheral blood lymphocytes of 37 normal healthy blood donors, with DNA from six tumor cell lines, or with 23 DNA samples prepared from various carcinoma tissues. To elucidate whether the integration of HERV-K genomes into the primate lineage occurred as a single event or as an integration with later expansion, we further examined the evolutionary history of HERV-K by Southern blot analyses with DNA samples from different primate species. We detected HERV-K genomes in Macaca mulatta and Macaca silenus, which represent Old World monkeys, but not in prosimians (Galago demidovii) and New World monkeys, represented by Saguinus fuscicollis, Saguinus oedipus, and Calliihrix iacchus. Thus, we assume that the infection of the primate lineage with HERV-K had occurred after the divergence of New World and Old World monkeys, but before the evolutionary expansion of large hominoids. In contrast to the apparent lack of HERV-Kenv sequences in DNA from tissue of the New World monkey Saguinus oedipus (cotton-top marmoset), we found HERV-K-DNA in the B95-8 cell-line, which is a Saguinus oedipus leukocyte cell-line, immortalized in vitro by Epstein-Barr virus (EBV) and cultivated in human cells. It may be speculated that HERV-K-DNA or HERV-K-particles were introduced into these cells during in vitro transformation with EBV.