Rachele Antonacci

Comparative mapping of human alphoid sequences in great apes using fluorescence in situ hybridization

Twenty-seven human alphoid DNA probes have been hybridized in situ to metaphase spreads of the common chimpanzee (PTR), the pigmy chimpanzee (PPA), and the gorilla (GGO) to investigate the evolutionary relationship between the centromeric regions of the great ape chromosomes. The surprising results showed that the vast majority of the probes did not recognize their corresponding homologous chromosomes. Alphoid sequences belonging to the suprachromosomal family 1 (chromosomes 1, 3, 5, 6, 7, 10, 12, 16, and 19) yielded very heterogeneous results: some probes gave intense signals, but always on nonhomologous chromosomes; others did not produce any hybridization signal. Almost all probes belonging to the suprachromosomal family 2 (chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22) recognized a single chromosome: chromosome 11 (phylogenetic IX) in PTR and PPA and chromosome 19 (phylogenetic V) in GGO. Localization of probes of suprachromosomal family 3 (chromosomes 1, 11, 17, and X) was found to be substantially conserved in PTR and PPA, but not in GGO. Probe pDMX1, specific for the human X chromosome, was the only sequence detecting its corresponding chromosome in all three species. PPA chromosomes I, IIp, IIq, IV, V, VI, and XVIII were never labeled, even under low-stringency hybridization conditions, by the 27 alphoid probes used in this study. These results, with particular reference to differences found in the two related species PTR and PPA, suggest that alphoid centromeric sequences underwent a very rapid evolution.

A panel of subchromosomal painting libraries representing over 300 regions of the human genome.

DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.

Evolution of TRG clusters in cattle and sheep genomes as drawn from the structural analysis of the Ovine TRG2@ locus

The availability of genomic clones representative of the T-cell receptor constant gamma (TRGC) ovine genes enabled us to demonstrate, by fluorescent in situ hybridization (FISH) on cattle and sheep metaphases, the presence of two T-cell receptor gamma (TRG1@ and TRG2@) paralogous loci separated by at least five chromosomal bands on chromosome 4. Only TRG1@ is included within a region of homology with human TRG locus on chromosome 7, thus TRG2@ locus appears to be peculiar to ruminants. The structure of the entire TRG2@ locus, the first complete physical map of any ruminant animal TCR gamma locus, is reported here. The TRG2@ spans about 90 kb and consists of three clusters that we named TRG6, TRG2, and TRG4, according to the constant genes name. Phylogenetic analysis has highlighted the correlation between the grouping pattern of cattle and sheep variable gamma (TRGV) genes and the relevant TRGC; variable (V), joining (J), and constant (C) rearrange to be found together in mature transcripts. The simultaneous results on the TRG2@ locus molecular organization in sheep and on the phylogenetic analysis of cattle and sheep V expressed sequences indicate that at least six TRG clusters distributed in the two loci are present in these ruminant animals. The inferred evolution of TRG clusters in cattle and sheep genomes is consistent with a scenario where a minimal ancient cluster, containing the basic structural scheme of one V, one J, and one C gene, has undergone a process of duplication and intrachromosomal transposition.

Artiodactyl emergence is accompanied by the birth of an extensive pool of diverse germline TRDV1 genes

Molecular cloning of cDNA from γ/δ T cells has shown that in sheep, the variable domain of the δ chain is chiefly determined by the expression of the TRDV1 subgroup, apparently composed of a large number of genes. There are three other TRDV subgroups, but these include only one gene each. To evaluate the extent and the complexity of the genomic TRDV repertoire, we screened a sheep liver genomic library from a single individual of the Altamurana breed and sheep fibroblast genomic DNA from a single individual of the Gentile di Puglia breed. We identified a total of 22 TRDV1 genes and the TRDV4 gene. A sequence comparison between germline and the rearranged genes indicates that, in sheep, the TRDV repertoire is generated by the VDJ rearrangement of at least 40 distinct TRDV1 genes. All germline TRDV1 genes present a high degree of similarity in their coding as well as in 5′ and 3′ flanking regions. However, a systematic analysis of the translation products reveals that these genes present a broadly different and specific repertoire in the complementarity-determining regions or recognition loops, allowing us to organize the TRDV genes into sets. We assume that selection processes operating at the level of ligand recognition have shaped the sheep TRDV germline repertoire. A phylogenetic study based on a sequence analysis of the TRDV genes from different mammalian species shows that the diversification level of these genes is higher in artiodactyl species compared to humans and mice.

Cloning and comparative mapping of recently evolved human chromosome 22-specific alpha satellite DNA.

We have isolated and characterized a new alphoid probe, named p190.22. Its chromosomal location was investigated using fluorescence in situ hybridization. Under high stringency conditions p190.22 recognizes specifically the centromere of chromosome 22. A chromosome 22-specific alphoid subset has been previously reported in the literature (p22/1∶2.1). The partial sequence and the genomic organization comparison strongly suggests that they recognize distinct subsets both specific for chromosome 22. The comparative mapping of probes p190.22 and p22/1∶2.1 on chimpanzee (PTR and PPA) and gorilla (GGO) chromosomes was investigated. The two probes showed different hybridization results. p190.22, in particular, did not show any hybridization signal in these three species, suggesting a recent evolution.

Comparative fluorescence in situ hybridization mapping of primate chromosomes with Alu polymerase chain reaction generated probes from human/rodent somatic cell hybrids

We have usedAlu polymerase chain reaction generated probes from rearranged human/rodent somatic cell hybrids for fluorescencein situ hybridization and comparative mapping of some intrachromosomal changes in the karyotypes of great apes (Pan troglodytes, P. paniscus, Gorilla gorilla Pongo pygmaeus), a gibbon (Hylobates lar), and an Old World monkey (Macaca fuscata). Probes containing chromosomes 2 and 18 fragments confirmed inversions already suggested by the banding pattern of great ape homologues. However, a chromosome 3 fragment showed complex rearrangements in the gibbon and macaque karyotype which were previously not well defined from banding. ‘Subchromosomal painting’ will allow the identification of intrachromosomal changes on the basis of DNA homology and provides a powerful method to study karyological and genomic evolution.

Generation of diversity by somatic mutation in the Camelus dromedarius T-cell receptor gamma variable domains

In jawed vertebrates the V‐(D)‐J rearrangement is the main mechanism generating limitless variations of antigen‐specific receptors, immunoglobulins (IGs), and T‐cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T‐cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V‐J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.

Expression and genomic analyses of Camelus dromedarius T cell receptor delta (TRD) genes reveal a variable domain repertoire enlargement due to CDR3 diversification and somatic mutation.

By a combination of rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR) we identified three T cell receptor delta variable (TRDV) subgroups and five joining (TRDJ) genes expressed in spleen, tonsils and blood of Camelus dromedarius. We provide evidence that the high diversity in sequence and length of the third complementarity determining region (CDR3) is a major component of the TR delta chain variability. Moreover, the identification of the corresponding germline genes allowed us to find out for the first time in a mammalian organism that productively rearranged TRDV genes undergo somatic mutation: the mutation rate of the analysed TRDV4 region is 0.013 per base pair in spleen and 0.009 in blood. The point mutations are scattered throughout the length of the variable domain from framework region FR1 to FR4. This random distribution of the amino acid changes, instead of its CDR clustering observed in immunoglobulins (IG), indicates that somatic mutation in dromedary, while contributing to the development of the TRDV repertoire, is not under antigen selection.

Molecular In Situ Hybridization Analysis of Sheep and Goat BAC Clones Identifies the Transcriptional Orientation of T Cell Receptor Gamma Genes on Chromosome 4 in Bovids

R. Antonacci1, G. Vaccarelli1, G.P. Di Meo2, B. Piccinni1, M.C. Miccoli1, E.P. Cribiu3, A. Perucatti2, L. Iannuzzi2 and S. Ciccarese1,∗ 1Department of Genetics and Microbiology, University of Bari, Bari, Italy; 2National Research Council (CNR), ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Naples, Italy; 3Departement de Genetique Animale, Centre de Recherche INRA de Jouy-en-Josas, France ∗Correspondence: E-mail: ciccarese@biologia.uniba.it

Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains

In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total of 331 mutations from a single animal, and comparing the properties of the mutation profiles of dromedary TRG and TRD variable domains. The transition preference and the significant mutation frequency in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lineages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rearrangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by the fact that our structural models, computed by adopting a comparative procedure, are consistent with the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural stability of the γδ subunits.