Synteny of human chromosomes 14 and 15 in the platyrrhines (Primates, Platyrrhini)

In order to study the intra- and interspecific variability of the 14/15 association in Platyrrhini, we analyzed 15 species from 13 genera, including species that had not been described yet. The DNA libraries of human chromosomes 14 and 15 were hybridized to metaphases of Alouatta guariba clamitans, A. caraya, A. sara, Ateles paniscus chamek, Lagothrix lagothricha, Brachyteles arachnoides, Saguinus midas midas, Leontopithecus chrysomelas, Callimico goeldii, Callithrix sp., Cebus apella, Aotus nigriceps, Cacajao melanocephalus, Chiropotes satanas and Callicebus caligatus. The 14/15 hybridization pattern was present in 13 species, but not in Alouatta sara that showed a 14/15/14 pattern and Aotus nigriceps that showed a 15/14/15/14 pattern. In the majority of the species, the HSA 14 homologue retained synteny for the entire chromosome, whereas the HSA 15 homologue displayed fragmented segments. Within primates, the New World monkeys represent the taxon with the highest variability in chromosome number (2n = 16 to 62). The presence of the HSA 14/15 association in all species and subspecies studied herein confirms that this association is the ancestral condition for platyrrhines and that this association has been retained in most platyrrhines, despite the occurrence of extensive inter- and intrachromosomal rearrangements in this infraorder of Primates.

Entropy analysis of DNA code dynamics in human chromosomes

Deoxyribonucleic acid, or DNA, is the most fundamental aspect of life but present day scientific knowledge has merely scratched the surface of the problem posed by its decoding. While experimental methods provide insightful clues, the adoption of analysis tools supported by the formalism of mathematics will lead to a systematic and solid build-up of knowledge. This paper studies human DNA from the perspective of system dynamics. By associating entropy and the Fourier transform, several global properties of the code are revealed. The fractional order characteristics emerge as a natural consequence of the information content. These properties constitute a small piece of scientific knowledge that will support further efforts towards the final aim of establishing a comprehensive theory of the phenomena involved in life.

Clastogenic effects of different Ureaplasma urealyticum serovars on human chromosomes

The possibility that Ureaplasma urealyticum might play an important role in human infertility was first raised more than 20 years ago, but this association remains speculative. Considering the hypothesis that the pathogenicity of Ureaplasma urealyticum may depend on its serotypes, the clastogenic effects of different strains of Ureaplasma urealyticum, at concentrations of 103 CCU (color changing units)/ml, 104 CCU/ml and 105 CCU/ml, were evaluated in vitro in short-term cultures of human lymphocytes. Total or partial mitotic inhibition was produced by Ureaplasma urealyticum serotypes 2, 3 and 10 independent of the concentration (103 CCU/ml, 104 CCU/ml or 105 CCU/ml) of the microorganisms employed. In contrast, the clastogenic effects observed with serotypes 1, 7 and 12 varied according to the concentration employed in the test. Mitotic alterations were observed in Ureaplasma urealyticum serotypes 5, 6, 7, 8, 9, 11 and 12. Chromatid gaps (53.0%) and chromatid breaks (13.9%) were the most frequent types of alterations observed. The results of this in vitro assay demonstrated that the clastogenic effects varied with the Ureaplasma urealyticum serotypes evaluated

RH maps of bovine chromosomes 15 and 29: conservation of human chromosomes 11 and 5

Comparative mapping data on evolutionary conserved coding sequences and synteny maps between human and cattle are insufficient to define the extent and distribution of conserved segments between these two species, because the order of loci is often rearranged. A 5000-rad cattle whole-genome radiation hybrid (WG-RH) panel was constructed to provide high-resolution comparative maps and also to integrate linkage maps of microsatellites with evolutionary conserved genes and transcripts in a single ordered map. We used the WG-RH panel to construct radiation hybrid maps of bovine Chromosomes (Chrs) 15 and 29 (BTA15 and BTA29), integrating microsatellites from published linkage maps with selected genes. The comprehensive map of BTA15 consists of 24 markers. 13 of which were placed in the framework map. Eleven molecular markers compose the comprehensive map of BTA29. seven of which were placed in the framework map. We identified the homologous regions between bovine Chr 15 (BTA15) and human Chrs 5 and 11 (HSA5 and HSA11), as well as between BTA29 and HSA11, the present study demonstrates that WG-RH mapping is an efficient method for integrating multiple genetic maps into one map and for incorporating monomorphic Type I loci into ordered maps for comparison between species.

THE MICROCELL-MEDIATED TRANSFER OF SINGLE HUMAN CHROMOSOMES INTO RECIPIENT MOUSE CELLS

Microcell-mediated chromosome transfer is a method of gene transfer which allows for the introduction of single or small groups of intact chromosomes into recipient host cells. Microcell transfer was first performed by Fournier and Ruddle using rodent microcells and various recipient cells. Expansion of this technology to include the transfer of normal human genetic material has been hindered because large micronucleate populations from diploid human cells have been unobtainable. This dissertation research describes, however, the methods for production of micronuclei in 40-60% of normal human fibroblasts. Once micronucleate cells were obtained, they were enucleated by centrifugation in the presence of Cytochalasin B; the microcells were then purified and fused to recipient mouse (LMTK('-)) cells using a new fusion protocol employing polyethylene glycol containing phytohemagglutinin. Microcell clones were isolated from the HAT selection system. Alkaline Giemsa staining performed on these hybrids indicated the presence of a single human chromosome in each of seven microcell clones from three separate experiments. That chromosome was further identified by G banding analysis to be human chromosome #17, which codes for thymidine kinase. The time course for production of these hybrids from fusion to karyotypic analysis was 6 weeks. The viability of the transferred human genetic material was assessed by electrophoretic isozyme analysis.^ Subsequent experiments were performed in an attempt to optimize the transfer frequency for the thymidine kinase gene using this system. Results indicated that the frequency could be increased from < 1 x 10('-6) in initial experiments to 2 x 10('-5) in the latest experiment. Analyses were also conducted to determine the number of chromosomes per isolated microcell as well as to investigate the stability of the transferred human chromosome in the mouse genome. ^

Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution

Chromosome painting in placental mammalians illustrates that genome evolution is marked by chromosomal synteny conservation and that the association of chromosomes 3 and 21 may be the largest widely conserved syntenic block known for mammals. We studied intrachromosomal rearrangements of the syntenic block 3/21 by using probes derived from chromosomal subregions with a resolution of up to 10–15 Mbp. We demonstrate that the rearrangements visualized by chromosome painting, mostly translocations, are only a fraction of the actual chromosomal changes that have occurred during evolution. The ancestral segment order for both primates and carnivores is still found in some species in both orders. From the ancestral primate/carnivore condition an inversion is needed to derive the pig homolog, and a fission of chromosome 21 and a pericentric inversion is needed to derive the Bornean orangutan condition. Two overlapping inversions in the chromosome 3 homolog then would lead to the chromosome form found in humans and African apes. This reconstruction of the origin of human chromosome 3 contrasts with the generally accepted scenario derived from chromosome banding in which it was proposed that only one pericentric inversion was needed. From the ancestral form for Old World primates (now found in the Bornean orangutan) a pericentric inversion and centromere shift leads to the chromosome ancestral for all Old World monkeys. Intrachromosomal rearrangements, as shown here, make up a set of potentially plentiful and informative markers that can be used for phylogenetic reconstruction and a more refined comparative mapping of the genome.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

Investigating the role of single point mutations in the human proteome: a computational study

In the post genomic era with the massive production of biological data the understanding of factors affecting protein stability is one of the most important and challenging tasks for highlighting the role of mutations in relation to human maladies. The problem is at the basis of what is referred to as molecular medicine with the underlying idea that pathologies can be detailed at a molecular level. To this purpose scientific efforts focus on characterising mutations that hamper protein functions and by these affect biological processes at the basis of cell physiology. New techniques have been developed with the aim of detailing single nucleotide polymorphisms (SNPs) at large in all the human chromosomes and by this information in specific databases are exponentially increasing. Eventually mutations that can be found at the DNA level, when occurring in transcribed regions may then lead to mutated proteins and this can be a serious medical problem, largely affecting the phenotype. Bioinformatics tools are urgently needed to cope with the flood of genomic data stored in database and in order to analyse the role of SNPs at the protein level. In principle several experimental and theoretical observations are suggesting that protein stability in the solvent-protein space is responsible of the correct protein functioning. Then mutations that are found disease related during DNA analysis are often assumed to perturb protein stability as well. However so far no extensive analysis at the proteome level has investigated whether this is the case. Also computationally methods have been developed to infer whether a mutation is disease related and independently whether it affects protein stability. Therefore whether the perturbation of protein stability is related to what it is routinely referred to as a disease is still a big question mark. In this work we have tried for the first time to explore the relation among mutations at the protein level and their relevance to diseases with a large-scale computational study of the data from different databases. To this aim in the first part of the thesis for each mutation type we have derived two probabilistic indices (for 141 out of 150 possible SNPs): the perturbing index (Pp), which indicates the probability that a given mutation effects protein stability considering all the “in vitro” thermodynamic data available and the disease index (Pd), which indicates the probability of a mutation to be disease related, given all the mutations that have been clinically associated so far. We find with a robust statistics that the two indexes correlate with the exception of all the mutations that are somatic cancer related. By this each mutation of the 150 can be coded by two values that allow a direct comparison with data base information. Furthermore we also implement computational methods that starting from the protein structure is suited to predict the effect of a mutation on protein stability and find that overpasses a set of other predictors performing the same task. The predictor is based on support vector machines and takes as input protein tertiary structures. We show that the predicted data well correlate with the data from the databases. All our efforts therefore add to the SNP annotation process and more importantly found the relationship among protein stability perturbation and the human variome leading to the diseasome.

Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromosome 18

PURPOSE: To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep. METHODS: Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia-anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites. RESULTS: The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations. CONCLUSIONS: This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

A single G-to-C change causes human centromere TGGAA repeats to fold back into hairpins.

Recently, we established that satellite III (TGGAA)n tandem repeats, which occur at the centromeres of human chromosomes, pair with themselves to form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. In separate studies, we have also established that the GCA triplet does not form bimolecular (GCA)2 motifs but instead promotes the formation of hairpins containing a GCA-turn motif in which the loop contains a single cytidine closed by a sheared G.A pair. Since TGCAA is the most frequent variant of TGGAA found in satellite III repeats, we reasoned that the potential of this variant to form GCA-turn miniloop fold-back structures might be an important factor in modulating the local structure in natural (TGGAA)n repeats. We report here the NMR-derived solution structure of the heptadecadeoxynucleotide (G)TGGAATGCAATGGAA(C) in which a central TGCAA pentamer is flanked by two TGGAA pentamers. This 17-mer forms a rather unusual and very stable hairpin structure containing eight base pairs in the stem, only four of which are Watson-Crick pairs, and a loop consisting of a single cytidine residue. The stem contains a (GGA)2 motif with intercalative 14G/4G stacking between two sheared G.A base pairs; the loop end of the stem consists of a sheared 8G.10A closing pair with the cytosine base of the 9C loop stacked on 8G. The remarkable stability of this unusual hairpin structure (Tm = 63 degrees C) suggests that it probably plays an important role in modulating the folding of satellite III (TGGAA)n repeats at the centromere.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Inheritance of the CENP-A chromatin domain is spatially and temporally constrained at human centromeres.

BACKGROUND: Chromatin containing the histone variant CENP-A (CEN chromatin) exists as an essential domain at every centromere and heritably marks the location of kinetochore assembly. The size of the CEN chromatin domain on alpha satellite DNA in humans has been shown to vary according to underlying array size. However, the average amount of CENP-A reported at human centromeres is largely consistent, implying the genomic extent of CENP-A chromatin domains more likely reflects variations in the number of CENP-A subdomains and/or the density of CENP-A nucleosomes within individual subdomains. Defining the organizational and spatial properties of CEN chromatin would provide insight into centromere inheritance via CENP-A loading in G1 and the dynamics of its distribution between mother and daughter strands during replication. RESULTS: Using a multi-color protein strategy to detect distinct pools of CENP-A over several cell cycles, we show that nascent CENP-A is equally distributed to sister centromeres. CENP-A distribution is independent of previous or subsequent cell cycles in that centromeres showing disproportionately distributed CENP-A in one cycle can equally divide CENP-A nucleosomes in the next cycle. Furthermore, we show using extended chromatin fibers that maintenance of the CENP-A chromatin domain is achieved by a cycle-specific oscillating pattern of new CENP-A nucleosomes next to existing CENP-A nucleosomes over multiple cell cycles. Finally, we demonstrate that the size of the CENP-A domain does not change throughout the cell cycle and is spatially fixed to a similar location within a given alpha satellite DNA array. CONCLUSIONS: We demonstrate that most human chromosomes share similar patterns of CENP-A loading and distribution and that centromere inheritance is achieved through specific placement of new CENP-A near existing CENP-A as assembly occurs each cell cycle. The loading pattern fixes the location and size of the CENP-A domain on individual chromosomes. These results suggest that spatial and temporal dynamics of CENP-A are important for maintaining centromere identity and genome stability.

Cytogenetic molecular delineation of a terminal 18q deletion suggesting neo-telomere formation

Deletion of the long arm of chromosome 18 is one of the most common segmental aneusomies compatible with life and usually involves a deletion of the terminal chromosomal region. However, the mechanisms implicated in the stabilization of terminal deletions are not well understood. In this study, we analyzed a girl with moderate mental retardation who had a cytogenetically visible terminal 18q deletion. In order to characterize the breakpoint in the terminal 18q region, we used fluorescence In situ hybridization (FISH) with bacterial artificial chromosomes (BACs) and pan-telomeric probes and also the array technique based on comparative genomic hybridization (array-CGH). FISH with pan-telomeric probes revealed no signal in the terminal region of the deleted chromosome, indicating the absence of normal telomere repeat (TTAGGG)n sequences in 18q. We suggest that neo-telomere formation by chromosome healing was involved in the repair and stabilization of this terminal deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.