A novel real-time PCR method for KIR genotyping.

Genes encoding killer cell immunoglobulin-like receptors (KIRs) are variable among individuals. Sequence-specific primer-directed polymerase chain reaction (PCR) amplification (PCR-SSP) and sequence-specific oligonucleotide hybridization of the PCR-amplified products (PCR-SSO) are the methods currently used to characterize the diversity of KIR gene content. Both these methods include time-consuming post-PCR analyses. Here, we developed a real-time PCR method that identifies the presence or absence of 16 KIR genes during PCR and avoids post-PCR analyses. This method is specific, sensitive, shortens the turnaround time compared with the conventional PCR-SSP and PCR-SSO methods, and it can be easily adapted for automation.

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. MATERIAL AND METHODS: Fibroblasts were stimulated for 48 h with 10(-4) or 10(-9) m Substance P; untreated fibroblasts served as controls. The expression of MMP-1, -2, -3, -7 and -11 and of TIMP-1 and -2 was evaluated using real-time polymerase chain reaction and western blotting. RESULTS: There was a significant, concentration-dependent stimulatory effect of Substance P on MMP-1, -2, -3 and -7 and TIMP-2 gene expression (p < 0.05), and a probable effect on MMP-11 (p = 0.06). At the higher concentration (10(-4) m Substance P), MMP-1, -2, -3, -7 and -11 and TIMP-2 showed the greatest up-regulation; at the lower concentration (10(-9) m Substance P), MMP-1, -3 and -7 and TIMP-2 exhibited diminished up-regulation, with MMP-2 and -11 showing down-regulation (p < 0.05). Expression of TIMP-1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up-regulated MMP-1, -2, -3 and -11 and TIMP-2. MMP-1, -3 and -11 and TIMP-2 showed greater up-regulation at the higher Substance P concentration and diminished up-regulation at the lower concentration. MMP-2 was up-regulated to a similar degree at both Substance P concentrations. CONCLUSION: In gingival fibroblast cells, Substance P at the higher concentration (10(-4) m) induced greater up-regulation of MMP-1, -3 and -11 and TIMP-2 expression, but at the lower concentration (10(-9) m) induced diminished up-regulation, which may represent a mechanism for modulating periodontal breakdown.

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.

Conserved organization of the ILT/LIR gene family within the polymorphic human leukocyte receptor complex.

The human leukocyte receptor complex (LRC) at Chromosome 19q13.4 encodes Ig superfamily proteins which regulate the function of various hematopoietic cell types. We investigated characteristics of the Ig-like transcript (ILT)/leukocyte Ig-like receptor (LIR) group of LRC genes in comparison with the other major LRC loci encoding the killer cell Ig-like receptors (KIRs). In direct contrast to KIR genes, the ILT/LIR loci of ethnically diverse individuals did not display haplotypic variations in gene number. Investigation of gene expression identified novel cDNA sequences related to the ILT2/LIR1, ILT4/LIR2, ILT3/LIR5, and ILT7 loci, while phylogenetic analysis revealed two distinct lineages of ILT/LIR genes. These two lineages differ in both the nature and extent of their sequence polymorphism. The presence of certain transcription factor-related motifs in the 5' untranslated region of ILT/LIR cDNAs correlates with the specific cell types in which particular ILT/LIR genes are expressed. Although extensive gene duplications and conversion events have apparently forged the LRC, our results indicate striking conservation in the organization of the ILT/LIR genes when compared with the related and closely linked KIR genes. This suggests the evolutionary maintenance of a significant function consistent with the cellular distribution of the ILT/LIR proteins.

Can molecular data place each neotropical monkey in its own branch?

Four different DNA datasets, representative of all extant neotropical primate genera, were tandemly aligned, comprising some 6,763 base pairs (bp) with 2,086 variable characters and 674 informative sites. Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analyses suggested three monophyletic families (Atelidae, Pitheciidae and Cebidae) that emerged almost at the same time during primate radiation. Combined molecular data showed congruent branching inside the atelid clade, placing Alouatta as the most basal lineage followed by Ateles and a more derived branch including Brachyteles and Lagothrix as sister groups. In the Pitheciidae, Callicebus was the most basal lineage with respect to Pithecia and to the more derived sister groups (Cacajao and Chiropotes). Conjoint analysis strongly supported the monophyly of the Cebidae, grouping Aotus, Cebus and Saimiri with the small callitrichines. Within callitrichines, Cebuella merged with Callithrix, Callimico appeared as a sister group of Callithrix/Cebuella, Leontopitecus as a sister group of the previous clade, and Saguinus was the earliest callitrichine offshoot. Two major points remained to be clarified in platyrrhine phylogeny: (i) the exact branching pattern of Aotus, Cebus, Saimiri and the callitrichines, and (ii), which two of these three families (Atelidae, Pitheciidae and Cebidae) are more closely related to one another.

Identification of seventeen novel KIR variants: fourteen of them from two non-Caucasian donors.

The killer-cell immunoglobulin-like receptors (KIR) expressed by human natural killer (NK) cells are encoded by a family of genes on chromosome 19. The number of KIR genes varies with haplotype and the individual genes exhibit polymorphism. To investigate KIR diversity we studied KIR cDNA and genes of four human donors: two Caucasians, one Black American and one Asian Indian. From analysis of these donors seventeen novel KIR variants were identified and characterized. Fifteen of the new variants appear to have a simple allelic relationship with a known KIR, whereas two of them combine the sequences of two different KIR genes. Fourteen of the seventeen KIR variants were isolated from the two non-Caucasoid blood donors. These data show that much human KIR diversity remains to be characterized, particularly in non-Caucasoid populations.

Gene assignment in Ateles paniscus chamek (Platyrrhini, Primates). Allocation of 18 markers of human syntenic groups 1, 2, 7, 14, 15, 17 and 22.

Eighteen markers allocated to human syntenic groups 1, 2, 7, 14, 15, 17 and 22 were assigned to the chromosome complement of the neotropical primate Ateles paniscus chamek. These new allocations and existing gene charts in this species were compared with chromosome painting patterns produced by human chromosome probes in the congeneric species A teles geoffroyi and with available data on the human genome and gene mapping. These comparisons showed congruent findings in Ateles and provided good evidence of how several human syntenic groups were evolutionarily rearranged.

Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans.

That NK cell receptors engage fast-evolving MHC class I ligands suggests that they, too, evolve rapidly. To test this hypothesis, the structure and class I specificity of chimpanzee KIR and CD94:NKG2 receptors were determined and compared to their human counterparts. The KIR families are divergent, with only three KIR conserved between chimpanzees and humans. By contrast, CD94:NKG2 receptors are conserved. Whereas receptors for polymorphic class I are divergent, those for nonpolymorphic class I are conserved. Although chimpanzee and human NK cells exhibit identical receptor specificities for MHC-C, they are mediated by nonorthologous KIR. These results demonstrate the rapid evolution of NK cell receptor systems and imply that "catching up" with class I is not the only force driving this evolution.

Mitochondrial DNA-like sequences in the nuclear genome of the opossum genus Didelphis (Marsupialia: Didelphidae).

This study reports the occurrence of mtDNA-like sequences in the nuclear genome of the opossum genus Didelphis (Didelphidae, Marsupialia). A specific primer pair designed to amplify a region encompassing a 3' terminal 118 bp region of the cytochrome b gene, the Thr and Pro tRNA genes, and a 489 bp region of the D-loop of the D. virginiana mtDNA, was used in highly stringent PCR reactions. These PCR reactions resulted in several fragments per individual varying in size from 259 bp to 1 kb. The sequencing of some of these fragments showed the occurrence of paralogous mtDNA-like sequences among the PCR amplified fragments. Analyses of qualitative aspects of these sequences, their transition/transversion ratios, and phylogenetic relationships were conclusive in showing the occurrence of mtDNA-like sequences in the nuclear genome of the genus Didelphis. Comparisons and phylogenetic analysis of orthologous mtDNA from the four Didelphis species and paralogous nuclear sequences suggested that mtDNA migration to the nuclear genome occurred more than once in Didelphis evolution.

Gene assignment in the spider monkey (Ateles paniscus chamek--APC): APE-MYH7 to 2q; AR-GLA-F8C to the X chromosome.

Comparative gene assignment between the spider monkey species Ateles paniscus chamek (APC) and man (HSA) showed conserved syntenic associations despite extensive karyotypic rearrangement between species. Two HSA 14q genes were allocated to APC 2q, being syntenic to other HSA 14q and HSA 15q markers previously assigned to APC 2q, and to HSA 12q genes previously assigned to APC 2p. These findings were consistent with A. geoffroyi chromosome painting with human whole-chromosome probes, indicating that the genus Ateles is karyotypically very rearranged. On the other hand, three human X-linked markers were assigned to the Ateles X chromosome, indicating that this chromosome is evolutionary stable.

Phylogenetic relationships of the Callitrichinae (Platyrrhini, primates) based on beta2-microglobulin DNA sequences.

The phylogenetic relationships of callitrichine primates have been determined by DNA sequence analyses of exons 1, 2, and 3 of the beta2-microglobulin gene. Parsimony, distance, and maximum likelihood analyses of ca. 900 base pairs of 21 taxa, representing all callitrichine genera, indicated that Saguinus was the most basal offshoot. Within Saguinus, S. fuscicollis appeared as the first divergent lineage followed by an unresolved trichotomy formed by S. mystax/S. imperator, S. midas/S. bicolor, and S. oedipus. A second callitrichine lineage was formed by Leontopithecus; each of the three species studied showed identical nucleotide sequences. Callimico appeared as the sister taxon of Callithrix/Cebuella. Genetic distances within this latter group were very small, although a stronger association between Cebuella and species of the Callithrix argentata group was observed. The inclusion of Cebuella in the genus Callithrix is suggested. These studies indicated that tamarins are more plesiomorphic than marmosets in agreement with the phyletic dwarfism hypothesis.

Molecular phylogeny of new world primates (Platyrrhini) based on beta2-microglobulin DNA sequences.

Neotropical primates, traditionally grouped in the infraorder Platyrrhini, comprise 16 extant genera. Cladistic analyses based on morphological characteristics and molecular data resulted in topologic arrangements depicting disparate phylogenetic relationships, indicating that the evolution of gross morphological characteristics and molecular traits is not necessarily congruent. Here we present a phylogenetic arrangement for all neotropical primate genera obtained from DNA sequence analyses of the beta2-microglobulin gene. Parsimony, distance, and maximum likelihood analyses favored two families, Atelidae and Cebidae, each containing 8 genera. Atelids were resolved into atelines and pitheciines. The well-supported ateline clade branched into alouattine (Alouatta) and ateline (Ateles, Lagothrix, Brachyteles) clades. In turn, within the Ateline clade, Lagothrix and Brachyteles were well-supported sister groups. The pitheciines branched into well-supported callicebine (Callicebus) and pitheciine (Pithecia, Cacajao, Chiropotes) clades. In turn, within the pitheciine clade, Cacajao and Chiropotes were well-supported sister groups. The cebids branched into callitrichine (Saguinus, Leontopithecus, Callimico, Callithrix-Cebuella), cebine (Cebus, Saimiri), and aotine (Aotus) clades. While the callitrichine clade and the groupings of species and genera within this clade were all well supported, the cebine clade received only modest support, and the position of Aotus could not be clearly established. Cladistic analyses favored the proposition of 15 rather than 16 extant genera by including Cebuella pygmaea in the genus Callithrix as the sister group of the Callithrix argentata species group. These analyses also favored the sister grouping of Callimico with Callithrix and then of Leontopithecus with the Callithrix-Callimico clade.

Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody.

Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the beta2-microglobulin (beta2m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two beta2m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of beta2m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of beta2m and residue 121 of the heavy chain. We examined the distribution of the two beta2m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of beta2m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of beta2m.

Evolutionary disruptions of human syntenic groups 3, 12, 14, and 15 in Ateles paniscus chamek (Platyrrhini, primates).

Comparative gene assignments of 18 markers, based on analyses of somatic cell hybrids and previous data in the literature, indicated that human (HSA) syntenic groups 3, 12, 14, and 15 are dissociated in the spider monkey species Ateles paniscus chamek (APC). Markers present in HSA 3p were allocated to APC 3 and APC 9. The HSA 12 cluster was split into two syntenic groups, one mainly including HSA 12p markers in APC 16 and the other, including HSA 12q markers, in APC 2p. The HSA 14q cluster split into three syntenic groups, corresponding to APC 2q, APC 6, and APC 12. Finally, the HSA 15 cluster split into two syntenic groups, APC 2q and APC 3. Comparisons with previous gene assignments and human SROs led to the tentative postulation of rearrangements having occurred during the evolutionary divergence of man and A. paniscus chamek. Chromosome painting data in the congeneric species A. geoffroyi, other New World and Old World primates, and several representative non-primate animals were compared in an attempt to delineate the ancestral and derived conditions underlying the evolutionary rearrangement of syntenic groups in mammals.

beta2-Microglobulin in neotropical primates (Platyrrhini).

Nucleotide sequences for the three exons of the beta2-microglobulin (beta2m) gene (B2m) were determined for 135 animals representing 37 species and all 16 genera of neotropical primates (Platyrrhini). Twenty-eight different nucleotide sequences, encoding for 26 different proteins, were obtained. In comparison with those of other primate species, the beta2-microglobulins of the Platyrrhini form a distinct clade. Individual genera of neotropical primates have distinctive B2m sequences, but within a genera species can have either the same or different B2m sequences. B2m polymorphism was found within three of the species sampled: Callicebus personatus, Saguinus midas, and Aotus azarae. Of these only the polymorphism in A. azarae has an effect upon the mature, functional beta2m protein: residue 4 being either alanine or threonine. The A. azarae B2m allele encoding alanine at position 4 is shared with another species of Aotus (A. infulatus). In pairwise comparison the mature beta2m proteins of neotropical primates differ by 1-9 amino acid substitutions which can occur at 18 positions within the sequence. The substitutions are distributed throughout the primary structure but are more commonly found in loops rather than beta strands of the tertiary structure. Of 17 residues of beta2m which hydrogen-bond with the class I heavy chain in human MHC class I molecules, 13 are conserved in the neotropical primates. The overall pattern of sequence variation in the B2m genes of the Platyrrhini is consistent with an evolution by successive selectively neutral events.

Comparative gene assignment in Ateles paniscus chamek (Platyrrhini, Primates) and man: association of three separate human syntenic groups and evolutionary considerations.

Regional assignment of eight markers to chromosome 2 of Ateles paniscus chamek (APC) confirmed a syntenic association similar to human (HSA) 12q + 14q + 15q. Three HSA 12q markers (RAP1B, PAH and ALDH2) were allocated to a shortest region of overlap (SRO) in APC 2p and found to be syntenic to other HSA 12q markers (PEPB and TCF1). Five HSA 14q markers (CTLA, PAX9, NSP, FOS and CHGA) were allocated to APC 2q and found to be syntenic to other HSA 14q markers (NP, TGM1, and CALM1) and to four HSA 15q markers (THBS1, B2M, HEXA and MPI) but dissociated from markers close to HSA 14qter (CKB) and HSA 15qter (FES-IDH2). Karyotypic comparisons showed an evident homoeology between APC 2p and HSA 12q while APC 2q was similar to an HSA 14qter::HSA 15qter fusion product. Comparative gene mapping data show that the HSA 14q + HSA 15q syntenic association is an ancestral mammalian gene cluster that has been maintained in several primate taxa. Conversely, in Ateles, it has been further associated with HSA 12q while, in Hominoids and Cebus, it has been independently dissociated into two separate syntenic groups, similar to HSA 14q and HSA 15q.

Comparative karyology in Akodon (Rodentia, Sigmodontinae) from Southeastern Brazil.

Karyotypic comparisons among five Akodon species showed that they shared a substantial proportion of their chromosome complements, indicated by distinct homologies between chromosomes, arms, or arm regions despite that this genus is karyotypically rearranged. A comparison with a related outgroup (Bolomys lasiurus) allowed for the recognition of ancestral and derived karyotypic traits as well as for inferring the direction of several chromosome rearrangements. This study indicated that species with lower diploid chromosome number are derived from species with higher diploid number, mainly by 11 fusions and 1 pericentric inversion while the direction of 5 other rearrangements could not be determined (fusions or fissions). UPGMA analyses of karyologic data indicated a topology like (Bolomys lasiurus (Akodon sp. (A. lindberghi (A. montensis (A. aff. cursor, A. cursor))))).

The human chromosome 3 gene cluster ACY1-CACNA1D-ZNF64-ATP2B2 is evolutionarily conserved in Ateles paniscus chamek (Platyrrhini, Primates).

Comparative mapping of Ateles paniscus chamek and man indicated that four human 3p markers are syntenic in this karyotypically rearranged neotropical primate. The evolutionary conservation of this gene cluster includes three adjacent human shortest regions of overlap (SROs): 3p21.1 (ACY1), 3p21.3-->p21.2 (CACNA1D), and 3p21.3 (ZNF64). A fourth syntenic marker (ATP2B2), at a more distal human SRO (3p26-->p25), indicated that human 3pter-->p14 is evolutionarily conserved in Ateles chromosome 3 (APC 3). Conversely, allocations of two human 3q markers (AGTR1 and IL12A) clearly excluded APC 3. Finally, allocation of the major histocompatibility complex class I genes further confirmed human 6p-6q dissociations in Ateles.

Assignment of TCF1, TGM1, CALM1, CKB, THBS1, B2M, and FES in Ateles paniscus chamek (Platyrrhini, Primates).

Regional assignment of five markers to chromosome 2 of Ateles paniscus chamek (APC) confirmed a syntenic association similar to human (HSA) 12q + 14q + 15q. TCF1 was allocated to a shortest region of overlap (SRO) in APC 2p and found to be syntenic to PEPB, while TGM1, CALM1, THBS1, and B2M were assigned to APC 2q, being syntenic to NP, HEXA, and MPI. Conversely, markers close to HSA 14qter (CKB) and HSA 15qter (FES-IDH2) were relocated to other Ateles syntenic groups. Karyotypic comparisons showed an evident homoeology between APC 2p and HSA 12q, whereas APC 2q was similar to an HSA 14qter::HSA 15qter fusion product.

Heteroduplex mobility assays (HMAs) and analogous sequence analysis of a cytochrome b region indicate phylogenetic relationships of selected callitrichids.

Heteroduplex mobility assays (HMAs) of a cytochrome b region were used for estimating genetic distances and phylogenetic relationships between some selected neotropical primates of the families Callitrichidae (marmosets and tamarins) and Cebidae [capuchin monkey, (Cebus apella)], and man (Homo sapiens). HMA distances were found to be strongly correlated to analogous estimates derived from DNA sequence data. Phylogenetic trees obtained by HMAs and sequence analyses showed similar topologies with almost identical intraspecific, intrageneric, and intergeneric relationships. The applicability of HMAs is assessed relative to different levels of molecular and organismal diversity.