Owl Monkey Alu Insertion Polymorphisms and Aotus Phylogenetics.

Owl monkeys (genus Aotus), or "night monkeys" are platyrrhine primates in the Aotidae family. Early taxonomy only recognized one species, Aotus trivirgatus, until 1983, when Hershkovitz proposed nine unique species designations, classified into red-necked and gray-necked species groups based predominately on pelage coloration. Recent studies questioned this conventional separation of the genus and proposed designations based on the geographical location of wild populations. Alu retrotransposons are a class of mobile element insertion (MEI) widely used to study primate phylogenetics. A scaffold-level genome assembly for one Aotus species, Aotus nancymaae [Anan_2.0], facilitated large-scale ascertainment of nearly 2000 young lineage-specific Alu insertions. This study provides candidate oligonucleotides for locus-specific PCR assays for over 1350 of these elements. For 314 Alu elements across four taxa with multiple specimens, PCR analyses identified 159 insertion polymorphisms, including 21 grouping A. nancymaae and Aotus azarae (red-necked species) as sister taxa, with Aotus vociferans and A. trivirgatus (gray-necked) being more basal. DNA sequencing identified five novel Alu elements from three different taxa. The Alu datasets reported in this study will assist in species identification and provide a valuable resource for Aotus phylogenetics, population genetics and conservation strategies when applied to wild populations.

BCL6 is regulated by p53 through a response element frequently disrupted in B-cell non-Hodgkin lymphoma.

The BCL6 transcriptional repressor mediates survival, proliferation, and differentiation blockade of B cells during the germinal-center reaction and is frequently misregulated in B-cell non-Hodgkin lymphoma (BNHL). The p53 tumor-suppressor gene is central to tumorigenesis. Microarray analysis identified BCL6 as a primary target of p53. The BCL6 intron 1 contains a region in which 3 types of genetic alterations are frequent in BNHL: chromosomal translocations, point mutations, and internal deletions. We therefore defined it as TMDR (translocations, mutations, and deletions region). The BCL6 gene contains a p53 response element (p53RE) residing within the TMDR. This p53RE contains a motif known to be preferentially targeted by somatic hypermutation. This p53RE is evolutionarily conserved only in primates. The p53 protein binds to this RE in vitro and in vivo. Reporter assays revealed that the BCL6 p53RE can confer p53-dependent transcriptional activation. BCL6 mRNA and protein levels increased after chemotherapy/radiotherapy in human but not in murine tissues. The increase in BCL6 mRNA levels was attenuated by the p53 inhibitor PFT-alpha. Thus, we define the BCL6 gene as a new p53 target, regulated through a RE frequently disrupted in BNHL.

Differential alu mobilization and polymorphism among the human and chimpanzee lineages.

Alu elements are primate-specific members of the SINE (short interspersed element) retroposon family, which comprise approximately 10% of the human genome. Here we report the first chromosomal-level comparison examining the Alu retroposition dynamics following the divergence of humans and chimpanzees. We find a twofold increase in Alu insertions in humans in comparison to the common chimpanzee (Pan troglodytes). The genomic diversity (polymorphism for presence or absence of the Alu insertion) associated with these inserts indicates that, analogous to recent nucleotide diversity studies, the level of chimpanzee Alu diversity is approximately 1.7 times higher than that of humans. Evolutionarily recent Alu subfamily structure differs markedly between the human and chimpanzee lineages, with the major human subfamilies remaining largely inactive in the chimpanzee lineage. We propose a population-based model to account for the observed fluctuation in Alu retroposition rates across primate taxa.

Y-chromosomal red-green opsin genes of nocturnal New World monkey.

The X-chromosomal locality of the red-green-sensitive opsin genes has been the norm for all mammals and is essential for color vision of higher primates. Owl monkeys (Aotus), a genus of New World monkeys, are the only nocturnal higher primates and are severely color-blind. We demonstrate that the owl monkeys possess extra red-green opsin genes on the Y-chromosome. The Y-linked opsin genes were found to be extremely varied, in one male appearing to be a functional gene and in other males to be multicopy pseudogenes. These Y-linked opsin genes should offer a rare opportunity to study the evolutionary fate of genes translocated to the Y chromosome.

Sequence and diversity of MHC DQA and DQB genes of the owl monkey Aotus nancymaae.

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates.

Analysis of constitutive heterochromatin of Aotus (Cebidae, Primates) by restriction enzyme and fluorochrome bands.

The current classification of genus Aotus includes nine species, four of which occur above the Amazon River and five below it. The position of several of these taxa as a valid species has been questioned. Recently, we described the chromosomal constitution of a population in the state of Rondonia, Brazil, whose karyotype typically presented a considerable accumulation of constitutive heterochromatin. To best characterize these heterochromatins, in this work we subjected the metaphases of these animals to banding using AluI, HaeIII, HinfI, RsaI, DdeI, MboI and MspI restriction enzymes and CMA3 and DAPI fluorochromes. The banded metaphases were also submitted to sequential C-banding. RsaI, DdeI and MboI enzymes showed, in all chromosomes, a banding pattern of C type, similar to that obtained using barium hydroxide. This banding was also seen with AluI, HinfI and MspI, but with reduction or elimination of the C-bands in the chromosome pairs 1, 3-7 and 9. MspI also reduced the C-band of pairs 11, 16-21 and 23. HaeIII induced intermediate bands between G and C. Considering the data of the different bands produced, it was possible to characterize at least three distinct types of constitutive heterochromatin in Aotus from Rondonia: (a) centromeric bands, (b) bands of the heterochromatic short arms and (c) interstitial bands.

Reverse transcriptase-polymerase chain reaction amplification and partial sequence of T helper 1- and T helper 2-type lymphokine genes from the owl monkey (Aotus trivirgatus).

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pair was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.

DNA evidence on the phylogenetic systematics of New World monkeys: support for the sister-grouping of Cebus and Saimiri from two unlinked nuclear genes.

Previous inferences from epsilon-globin gene sequences on cladistic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were tested by strength of grouping and bootstrap values for the clades in the most parsimonious trees found: for this epsilon data set enlarged with additional Cebus and Saimiri orthologues; for another nuclear DNA sequence data set consisting of IRBP (interstitial retinol-binding protein gene) intron 1 orthologues; and for tandemly combined epsilon and IRBP sequences. Different ceboid species of the same genus always grouped strongly together as demonstrated by results on Cebus (capuchin monkeys), Saimiri (squirrel monkeys), Callicebus (titi monkeys), Aotus (night monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys). Other strong groupings that could be represented as monophyletic taxa in a cladistic classification were: Cebuella (pygmy marmoset) and Callithrix (marmoset) into subtribe Callitrichina; Callitrichina, Callimico (Goeldi's monkey), Leontopithecus (lion tamarin), and Saguinus (tamarin) into subfamily Callitrichinae; Callitrichinae, Aotus, Cebus, and Saimiri into family Cebidae; Cacajao (uakari monkey) and Chiropotes (saki) into subtribe Chiropotina; Chiropotina and Pithecia (bearded saki) into tribe Pitheciini; Pitheciini and Callicebus into subfamily Pitheciinae; Brachyteles (woolly spider monkey), Lagothrix (woolly monkey), and Ateles into tribe Atelini; and Atelini and Alouatta into subfamily Atelinae. In addition the epsilon and IRBP results congruently grouped (but at lesser strengths) Brachyteles and Lagothrix into subtribe Brachytelina within Atelini, and also Cebus and Saimiri into subfamily Cebinae within Cebidae. Because the IRBP results weakly grouped Pitheciinae with Cebidae, whereas the epsilon results weakly grouped Pitheciinae with Atelinae, the present evidence is best represented in an interim cladistic classification of ceboids by dividing the superfamily Ceboidea into three families: Atelidae, Pitheciidae, and Cebidae.

The coding sequence of Duffy blood group gene in humans and simians: restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy-negative individuals.

The coding and untranslated flanking sequences of Duffy gene (FY) in humans and simians are in a single exon. The difference between the two codominant alleles, FY*A and FY*B, is a single change at nucleotide 306: guanidine is in FY*A and adenine is in FY*B. This produces a codon change that subsequently modifies the amino acid at position 43 of gpFy, the major subunit of the Duffy blood group protein complex. The glycine at this position in antigen Fya exchanges with aspartic acid in antigen Fyb. The guanidine at nucleotide 306 creates an additional Ban I restriction site in FY*A. Ban I digestion of DNA-PCR amplified products of FY*B and FY*A yields three and four fragments, respectively. Restriction fragment length polymorphism (RFLP) studies show that Fy(a+b-) and Fy(a-b+) whites are FY homozygous, that most Fy(a-b-) blacks have FY*B, and most Fy(a+b-) blacks are FY*A/FY*B heterozygous. In the black population a silent FY*B is very common, but a silent FY*A has not been found yet. On RNA blot analysis, the gpFy cDNA clone detected mRNA in the lung, spleen, and colon but not in the bone marrow of Duffy-negative individuals. Therefore, there is no null phenotype in Fy(a-b-) blacks. The gpFy homology between human and chimpanzee is 99% with a single residue change at position 116 (valine to isoleucine), whereas a 94% homology is found in squirrel and rhesus monkeys, and there is a 93% homology in aotus monkey when compared with humans. The N-terminal exocellular domain of simian gpFy helps to identify a set of amino acids critical for antibody and malarial parasite specificities.

Conservation of the HLA-DQB2 locus in nonhuman primates.

The evolutionary history of MHC class II genes is characterized by several examples of gene duplication, leading both to the creation of distinct subregions such as DR, DQ, and DP, as well as to duplicated loci within each of these subregions. In the human MHC, a prominent example of this diversification occurs within the HLA-DQ subregion, where the nonpolymorphic and transcriptionally "silent" DQB2 locus is highly homologous to the polymorphic expressed DQB1 locus. In order to gain some insight into the mechanisms constraining polymorphism at the DQB2 locus, the second exons of five nonhuman primate DQB2 alleles were sequenced. Six nonhuman primate DQB2 analogous sequences were obtained, two each from chimpanzee and owl monkey cell lines, and one each from gorilla and gibbon cell lines. Notably, the DQB2 sequences from the gibbon, gorilla, and one of the two chimpanzee sequences, although containing some silent nucleotide changes, encode a predicted DQB2 protein with 100% homology to the human DQB2 sequence. The owl monkey DQB2 allelic sequences and the other chimpanzee DQB2 sequence contain additional polymorphisms, but maintain approximately 95% nucleotide sequence identity with human and the other primate sequences. Identification of the owl monkey DQB2 locus indicates that the ancestral DQ gene duplication event occurred at least 40 million years ago, rather than 10 million years, as previously thought. Remarkably, nucleotide sequences from amplified cDNA indicate that the DQB2 gene, and not the DQB1 gene, may be transcribed in the owl monkey line. Substitutions occur at sites comparable to codons of well-recognized allelic variation in the functional DQB1 genes, implying that variation within the DQB2 locus operates under similar selection constraints to the DQB1 locus, with an extremely high degree of conservation through primate evolution.

Twinning in the karyotype I night monkey (Aotus nancymai).

A case of successful survival of twins in a Karyotype I (2n = 54) night monkey (Aotus nancymai) is described. Monthly weights of the pregnant female, and of the twins, are compared to those of pregnant females and their single birth offspring of the same sex and karyotype.

Genetic linkage analysis and homology relationships of genes located on human chromosome 11q.

We have used DNA polymorphisms detected by probes for 11q to order 16 genes and to determine the genetic distances between them. Our map includes the genes for CD20, tyrosinase, progesterone receptor, stromelysin, collagenase, N-CAM, dopamine-D2 receptor, apolipoproteins AI-CIII-AIV, CD3-epsilon, -delta, and -gamma, porphobilinogen deaminase, thy-1, and ets-1. These genes have previously been sequenced as well as placed on the 11q cytogenetic map, which now makes them anchor points between the cytogenetic, genetic, and physical maps of this region. The ordering and distances between these genes are of immediate use in testing hypotheses of candidate genes for human genetic diseases associated with chromosome 11q. A comparison between our genetic map and similar maps from other species defines regions of homologous synteny that may be useful in mapping human genetic disease genes localized to the 11q region. Analysis of such homology provides additional bases for speculation of the evolutionary histories of gene families in this region.

Homologs of eleven loci on human chromosome 11 assigned to two owl monkey chromosomes.

We localized 11 loci mapped to human chromosome 11 to two chromosomes, 4 and 19 of owl monkey karyotype VI (2n = 49/50), by the use of somatic cell hybrids. Furthermore, using in situ hybridization to chromosomes of two owl monkey karyotypes, the HSTF1 oncogene locus was precisely localized on homologs 19q (K-VI) and 2q (K-II). Comparative analysis of available gene-mapping data among human, mouse, and owl monkey chromosomes revealed a pattern of evolutionary change in a syntenic group on human chromosome 11. These structural changes could be explained as having derived from a pericentric inversion of human chromosome region 11cen----q13 and a translocation involving human region 11q22----qter during primate evolution.

The involucrin genes of pig and dog: comparison of their segments of repeats with those of prosimians and higher primates.

The involucrin genes of the dog and the pig have been cloned and sequenced. Like the corresponding genes of the prosimians, each contains a homologous segment of short tandem repeats at the same position in the coding region. However, the codon sequence of the repeats in the prosimians differs significantly from that of the nonprimate mammals. This evolution has been brought about by a combination of genetic modifications (selective deletions, mutations, and gene conversions). In the anthropoids, this segment of repeats was replaced by a modern one differing in location, sequence, and repeat length. In several of its properties the modern segment has continued the prosimian trend away from the nonprimates. The overall direction of the evolution of this segment has therefore been maintained even though there have been sudden changes in the evolutionary processes acting on the gene.

Owl monkey gene map: evidence for a homologous human chromosome 7q region near the cystic fibrosis locus.

We have demonstrated the assignments of two gene loci (COLIA2, MET) and two noncoding DNA markers (D7S13, D7S8) to owl monkey chromosome 14 (K-VI) by hybridizing DNA probes from the cystic fibrosis (CF) region of human chromosome 7q21-32 to panels of rodent-owl monkey somatic cell hybrids. The assignments are substantiated by in situ chromosome hybridization of markers COLIA2, MET, and D7S13 to the distal long arm of chromosome 14 (K-VI). These results support genomic conservation of the human CF region, at least in the higher primates.

The involucrin gene of the owl monkey: origin of the early region.

A large part of the coding region of the hominoid involucrin gene is of recent origin. This part of the gene, which we have called the modern segment, contains numerous repeats of a sequence of 10 codons, created by multiple duplications some of which consist of 3-12 repeats. We have sequenced two alleles of the involucrin gene in the owl monkey and found that the involucrin gene of this species also possesses a modern segment. By comparing the modern segment of the owl monkey with that of the hominoids, we find that only a part of this segment is shared by the two species. We call this part the early region because it must have originated in a common ancestor of the anthropoids. The rest of the hominoid modern segment does not correspond to any groups of repeats in the owl monkey and was therefore created after divergence of the two lineages. As in the hominoids, the latest additions to the modern segment of the owl monkey have been in its 5' half, which possesses different duplication patterns in the two alleles. Lineage divergences within the anthropoids can be detected at different sites within the modern segment.

Molecular evidence of Y-autosomal translocations in owl monkeys.

Probe pDP1007, which contains highly conserved DNA sequences from the sex-determining region of the human Y chromosome, cross-hybridized with owl monkey EcoRI restriction fragments of 1.8 kb and 6.6 kb. Southern transfer analysis of owl monkey (karyotype VI)--rodent somatic cell hybrids localized the 1.8-kb fragment on the owl monkey X chromosome and the 6.6-kb fragment, which is male specific, on chromosome 14/Y. Regional in situ chromosome mapping of pDP1007 revealed specific sites of hybridization: the distal short arm of the X chromosome of karyotypes IV, VI, and VII; the small metacentric Y of karyotype IV; the C-band positive region on the short arm of chromosome 17/Y (karyotype VII); and the C-band positive region on the long arm of chromosome 14/Y (karyotype VI). These molecular findings reinforce cytological evidence that Y-chromosomal material has been transferred to autosomes 14 and 17 in owl monkeys of karyotypes VI and VII, respectively, in which there are no independently segregating Y chromosomes.

A putative homoeolog of human chromosome 12 in the owl monkey.

Analyses of Southern blots of rodent x owl monkey somatic cell hybrids permitted syntenic assignment of gene loci coding for triosephosphate isomerase (TPI), antigen CD4(T4), Kirsten rat sarcoma 2(KRAS2) virus, insulin-like growth factor 1 (IGF1), and alpha 2-macroglobulin (A2M) to chromosome 10 of owl monkey karyotype VI(2n = 49, 50). In addition, regional in situ localization of the T4 and KRAS2 loci on the proximal region of the long arm of this acrocentric chromosome and on the corresponding homologous region on the long arm of metacentric chromosome 1 of karyotype IV (2n = 52) substantiated our hypothesis that a single fusion or fission event is responsible for the polymorphism in chromosome number characteristic of owl monkeys from at least three allopatric populations. The study supports a putative homoeology between owl monkey chromosome 10 (K-VI) and human chromosome 12. The morphological differences between these two primate chromosomes indicate evolutionary rearrangements involving at least one pericentric inversion.

Chromosome assignment of the owl monkey MOS and MYC gene loci.

Southern blot hybridizations of rodent x owl monkey hybrid DNAs with human cDNA probes allowed the mapping of the MOS and MYC gene loci to owl monkey chromosome 16 of karyotype VI (2n = 49 male/50 female) and to the homologous chromosome 15 of karyotype V (2n = 46). Synteny of MOS and MYC gene loci in both man and owl monkey suggests this chromosome segment's conservation in primates, contrasting with its disruption in the mouse.

Chromosome assignment of the gene loci ETS1 and THY1 in the owl monkey.

Our previous assignment of the gene loci HBB, HRAS1, INS, PTH, LDHA, and CAT to owl monkey chromosome 19 of karyotype VI (K-VI) indicated a putative homology of this owl monkey chromosome with the short arm of human chromosome 11 (HSA 11p). To investigate further the extent of shared homology, we localized in the owl monkey complement two genes known to be on HSA 11q. Segregation analysis of ETS1 and THY1 homologous DNA in three karyotypically different panels of rodent x owl monkey somatic cell hybrids provided evidence for the syntenic assignment of these loci to homologous chromosomes of three owl monkey karyotypes, namely, chromosomes 4 (K-VI), 3 (K-II), and 5 (K-V). The results indicate a disruption of syntenic gene loci on the distal portion of HSA 11q from 11p during primate evolution.