Alphoid repetitive DNA in human chromosomes

The thesis describes the first extensive DNA sequence analysis that demonstrated that the tandemly repeated alphoid DNA in the centromere of the human chromosomes consists of distinct subfamilies and in a number equal to or exceeding the number of chromosomes. The expected presence of only one or a few distinct subfamily on individual chromosomes was supported by the characterization of an extremely well-defined subfamily specific for chromosome 7 and represented in the original collection of subfamilies. The pattern of chromosome-specificity breaks down among the acrocentric chromosomes where chromosomes 13 and 21 were found to share one and chromosomes 14 and 22 to share another specific subfamily. By in situ hybridization these subfamilies were shown not to be shared by other chromosomes. The remarkable pairwise pattern of sequence homogenization was present also in the chimpanzee genome raising the question of its biological role. However, the subfamilies on these human and chimpanzee chromosomes are not orthologous but were shown to originate from two evolutionarily different repeat families. It follows that dramatic sequence evolution has occurred in one or both species during or after separation. The sequence evolution might even occur at a higher rate in humans. This possibility was studied in orthologous alphoid sequences on the X chromosome of humans and the great apes. The analysis supports the general view that our closest relative is the chimpanzee and indicates that the rate of recombination is increased in the human repeat DNA. A "molecular clock" running faster in this DNA may have evolutionary implications. Finally, the usefulness of alphoid subfamilies as chromosome-specific markers is illustrated in a cytogenetic dissection of the centromeric region of Robertsonian translocations. The breakpoints were located to satellite III DNA leaving these chromosomes dicentric. The order of the different tandem DNAs on the p-arm of the acrocentric chromosomes could also be established.     

Interspecies comparative genome hybridization and interspecies representational difference analysis reveal gross DNA differences between humans and great apes

Comparative chromosome G-/R-banding, comparative gene mapping and chromosome painting techniques have demonstrated that only few chromosomal rearrangements occurred during great ape and human evolution. Interspecies comparative genome hybridization (CGH), used here in this study, between human, gorilla and pygmy chimpanzee revealed species-specific regions in all three species. In contrast to the human, a far more complex distribution of species-specific blocks was detected with CGH in gorilla and pygmy chimpanzee. Most of these blocks coincide with already described heterochromatic regions on gorilla and chimpanzee chromosomes. Representational difference analysis (RDA) was used to subtract the complex genome of gorilla against human in order to enrich gorilla-specific DNA sequences. Gorilla-specific clones isolated with this technique revealed a 32-bp repeat unit. These clones were mapped by fluorescence in situ hybridization (FISH) to the telomeric regions of gorilla chromosomes that had been shown by interspecies CGH to contain species-specific sequences.     

Commentary: tensions between the theory and practice of palliative care

Structural variations between great ape and human chromosomes due to pericentric inversions and translocations have created at apparent controversy during the reconstruction of hominoid phylogeny. One such variation involves human chromosome 5, which is equivalent to chromosome 4 in chimpanzee and orangutan but equivalent to segments of chromosomes 4 and 19 in gorilla. Obviously, neither banding patterns nor centromeric indecies in these chromosomes match. The pathological condition of cri du chat syndrome is due to the cytogenetic deletion of band p15.2 of chromosome 5. Is this region involved during pericentric inversion of apes chromosome 4? We used a human cosmid probe for cri du chat syndrome as a phylogenetic marker in search of the aforementioned question. The genomic sequences for cri du chat syndrome region were conserved in chimpanzee (PTR4) and orangutan (PPY4) but displayed a positional divergence in gorilla on chromosome 19(GG019). In addition, we used a human cosmid DNA probe for DiGeorge syndrome which is located on chromosome 22 band q11.2 and was conserved within band 23q11.2 in apes. The loci specific human genomic probes may help to describe the inversions and translocations for other chromosomes.     

Bacterial flora in chronic inner ear infections in adults

The human genome carries multiple copies of sequences related to endogenous retroviral DNA. We report here the distribution of a new multicopy long terminal repeat (LTR) sequence that has been a part of an endogenous retrovirus-like sequence RTVL-H2. Twenty-four human chromosomes were either separated by flow sorting or by using rodent cells carrying a single human chromosome, and the DNA was subjected to Southern analyses using the RTVL-H2 DNA as a probe. The RTVL-H2 LTRs were distributed among all the human chromosomes, but the density and the profile differed from chromosome to chromosome. The same chromosome obtained from different individuals showed essentially the same chromosome-specific patterns. The distribution of the RTVL-H2 LTRs among different chromosomes did not correlate with the distribution of LTRs from another endogenous retroviral DNA, HERV-A, strongly suggesting that there is no preferred chromosome or a region thereof, for the integration. The possibility of rearrangement or amplification after integration is discussed.     

Tiggers and DNA transposon fossils in the human genome

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.     

Analysis of on- and off-resonance magnetization transfer techniques

PURPOSE: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. METHODS AND MATERIALS: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. RESULTS: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. CONCLUSIONS: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1 (translocations) were readily scored and found to equate with the complementary asymmetrical exchanges (dicentrics). That is, nonlethal stable changes, which might be of concern in carcinogenic processes, complement lethal, unstable changes. Interstitial deletions that may contribute to the loss of antioncogenes as well as to lethality are also readily detected with enhanced levels detected at higher doses. The high level of induced terminal deletions and of incomplete dicentrics and translocations indicate a partial failure of interaction between lesions induced in human and hamster DNA, and suggest that such interspecies interactions lack the fidelity of intraspecies DNA lesion interactions. This suggests caution in the use of such model systems as indicators of human cell responsiveness.     

Molecular characterization of a novel repetitive element from Pneumocystis carinii from rats

A repetitive DNA sequence, Rp-alpha, was isolated from rat-derived Pneumocystis carinii. The genome of rat-derived P. carinii contained 10 to 15 copies of Rp-alpha, which were located on most chromosomes, but no Rp-alpha could be detected in P. carinii derived from either humans or mice. Sequence analysis of two copies of the repeat showed them to be related but distinct. Each of them contained several copies of the 9-base sequence TAACCCTAA, arranged as direct repeats. Oligonucleotides consisting of multimers of this 9mer hybridized to the same set of chromosomes recognized by cloned copies of the Rp-alpha repeat. When used in DNA fingerprinting, the Rp-alpha repeat was capable of distinguishing between different isolates of rat-derived P. carinii.     

Inverted repetitive sequences in the human genome

A specific class of DNA sequences, the inverted repetitive sequences, forms hairpin-like structures in denatured DNA by the folding back of a single linear chain. The reassociation process of these sequences is unimolecular and the rate is extremely fast. Inverted repetitive sequences comprise 6% of the total human genome. They appear to be heterogeneous in length with an overall average length of 190 nucleotides. The inverted sequences are represented in almost all families of repetition frequencies, highly repetitive as well as very few copies per genome. They are not localized at unique sites on metaphase chromosomes. It is estimated that there are approximately 2 X 10(6) inverted repeats per haploid human genome. The biological function of this class of sequences is unknown.     

Reciprocal chromosome painting between human and prosimians (Eulemur macaco macaco and E. fulvus mayottensis)

We used fluorescence in situ hybridisation to delineate the homology between the human karyotype and those of two lemur species (Eulemur macaco macaco and E. fulvus mayottensis). Human and lemur chromosome-specific probes were established by bivariate fluorescence-activated flow sorting (FACS) and subsequent degenerate oligonucleotide-primed PCR (DOP-PCR). Reciprocal painting of human probes to lemur chromosomes and vice versa allowed a detailed analysis of the interchromosomal rearrangements that had occurred during the evolution of these species. The results indicate that the genomes of both species have undergone only a few translocations during more that 45 million years of lemur and human evolution. The synteny of homologs to human chromosomes 3, 9, 11, 13, 14, 17, 18, 20, 21, X, and Y was found to be conserved in the two lemur species. Taking non-primate mammals as the outgroup for primates, ancestral conditions for various primate chromosomes were identified and distinguished from derived forms. Lemur chromosome painting probes were also used for cross-species hybridization between the two lemur species. The results support an earlier assumption, made on the basis of chromosome banding, that the karyotypes of the two species have evolved exclusively by Robertsonian transformations. All probes derived from E. f. mayottensis chromosomes specific for homologs involved in rearrangements in E. m. macaco exclusively painted entire chromosome arms. The results further indicate that E. f. mayottensis most probably has a more ancestral karyotype than E. m. macaco. Probes derived from prosimians will be useful in comparing the karyotypes of other lower primates, which will improve our understanding of early primate genome evolution.     

Members of the Arabidopsis actin gene family are widely dispersed in the genome

Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number. The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes. Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation. All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes. The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5). Even the members of three closely related and recently duplicated pairs of actin genes are unlinked. Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evolutionary history. Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.     

Oat-maize chromosome addition lines: a new system for mapping the maize genome

Novel plants with individual maize chromosomes added to a complete oat genome have been recovered via embryo rescue from oat (Avena sativa L., 2n = 6x = 42) x maize (Zea mays L., 2n = 20) crosses. An oat-maize disomic addition line possessing 21 pairs of oat chromosomes and one maize chromosome 9 pair was used to construct a cosmid library. A multiprobe (mixture of labeled fragments used as a probe) of highly repetitive maize-specific sequences was used to selectively isolate cosmid clones containing maize genomic DNA. Hybridization of individual maize cosmid clones or their subcloned fragments to maize and oat genomic DNA revealed that most high, middle, or low copy number DNA sequences are maize-specific. Such DNA markers allow the identification of maize genomic DNA in an oat genomic background. Chimeric cosmid clones were not found; apparently, significant exchanges of genetic material had not occurred between the maize-addition chromosome and the oat genome in these novel plants or in the cloning process. About 95% of clones selected at random from a maize genomic cosmid library could be detected by the multiprobe. The ability to selectively detect maize sequences in an oat background enables us to consider oat as a host for the cloning of specific maize chromosomes or maize chromosome segments. Introgressing maize chromosome segments into the oat genome via irradiation should allow the construction of a library of overlapping fragments for each maize chromosome to be used for developing a physical map of the maize genome.     

Two tandemly repeated telomere-associated sequences in Nicotiana plumbaginifolia

Two tandemly repeated telomere-associated sequences, NP3R and NP4R, have been isolated from Nicotiana plumbaginifolia. The length of a repeating unit for NP3R and NP4R is 165 and 180 nucleotides respectively. The abundance of NP3R, NP4R and telomeric repeats is, respectively, 8.4 x 10(4), 6 x 10(3) and 1.5 x 10(6) copies per haploid genome of N. plumbaginifolia. Fluorescence in situ hybridization revealed that NP3R is located at the ends and/or in interstitial regions of all 10 chromosomes and NP4R on the terminal regions of three chromosomes in the haploid genome of N. plumbaginifolia. Sequence homology search revealed that not only are NP3R and NP4R homologous to HRS60 and GRS, respectively, two tandem repeats isolated from N. tabacum, but that NP3R and NP4R are also related to each other, suggesting that they originated from a common ancestral sequence. The role of these repeated sequences in chromosome healing is discussed based on the observation that two to three copies of a telomere-similar sequence were present in each repeating unit of NP3R and NP4R.     

Pulmonary function values in Friesian and double-muscled calves during acute poikilocapnic hypoxia

In the genus Eulemur (Malagasy lemurs) karyotype diversification has occurred mainly through Robertsonian mechanisms of chromosome fusion (Rumpler et al., 1976). Eulemur coronatus is the sole species to have the largest genome size, due to a very large amount of C-heterochromatin, mostly located at the pericentromeric regions of the largest chromosomes (Warter and Rumpler, 1985). This increase in C-heterochromatin was thought to be due to DNA amplification (Ronchetti et al., 1993). The aim of this work was to investigate whether the large C-heterochromatin of Eulemur coronatus might have derived by amplification of the smaller C-heterochromatin of Eulemur macaco, a closely related species with smaller genome size. To obtain information on the overall base composition of the total genomes, on the relative interspersion of AT and GC base paris along the DNA molecule and on the structural differences in C-heterochromatin, we used a quantitative cyto-chemical approach, based on fluorescence resonance energy transfer (FRET) between DNA-specific fluorochromes (i.e. the AT-specific Hoechst 33258, and the non base-specific dye, propidium iodide). Micro-spectrofluorometry and image analysis were used to investigate both the overall FRET efficiency and its spatial distribution along the chromosome arms. FRET efficiency values of the DNA in C-heterochromatin were significantly different in the two Eulemur species, indicating a different qualitative composition of repetitive DNA. This suggests that the repetitive DNA of Eulemur coronatus cannot have originated by amplification in toto of the repetitive DNA sequences of Eulemur macaco.     

Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line.     

Chromosome mapping of low-temperature induced Wcs120 family genes and regulation of cold-tolerance expression in wheat

Low-temperature (LT) induced genes of the Wcs120 family in wheat (Triticum aestivum) were mapped to specific chromosome arms using Western and Southern blot analysis on the ditelocentric series in the cultivar Chinese Spring (CS). Identified genes were located on the long arms of the homoeologous group 6 chromosomes of all 3 genomes (A, B, and D) of hexaploid wheat. Related species carrying either the A, D, or AB genomes were also examined using Southern and Western analysis with the Wcs120 probe and the WCS120 antibody. All closely related species carrying one or more of the genomes of hexaploid wheat produced a 50 kDa protein that was identified by the antibody, and a Wcs120 homoeologue was detected by Southern analysis in all species. In the absence of chromosome arm 6DL in hexaploid CS wheat no 50 kDa protein was produced and the high-intensity Wcs120 band was missing, indicating 6DL as the location of Wcs120 but suggesting silencing of the Wcs120 homoeologue in the A genome. Levels of proteins that cross-reacted with the Wcs120 antibody and degrees of cold tolerance were also investigated in the Chinese Spring/Cheyenne (CS/CNN) chromosome substitution series. CNN chromosome 5A increased the cold tolerance of CS wheat. Densitometry scanning of Western blots to determine protein levels showed that the group 5 chromosome 5A had a regulatory effect on the expression of the Wcs120 gene family located on the group 6 chromosomes of all three hexaploid wheat genomes.     

A bacterial artificial chromosome-based framework contig map of human chromosome 22q

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.