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PubMed2003

DNA helix: the importance of being GC-rich.

A new explanation for the emergence of heavy (GC-rich) isochores is proposed, based on the study of thermostability, bendability, ability to B-Z transition and curvature of the DNA helix. The absolute values of thermostability, bendability and ability to B-Z transition correlated positively with GC content, whereas curvature correlated negatively. The relative values of these parameters were determined as compared to randomized sequences. In genes and intergenic spacers of warm-blooded animals, both the relative bendability and ability to B-Z transition increased with elevation of GC content, whereas the relative thermostability and curvature decreased. The usage of synonymous codons in GC-rich genes was also found to augment bendability and ability to B-Z transition and to reduce thermostability of DNA (as compared to synonymous codons with the same GC content). The analysis of transposable elements (Alu and B2 repeats in the human and mouse) showed that the level of their divergence from the consensus sequence positively correlated with relative bendability and ability to B-Z transition and negatively with relative thermostability. The bendability and ability to B-Z transition are known to relate to open chromatin and active transcription, whereas curvature facilitates chromatin condensation. Because heavy isochores are known to be gene-rich and show a high level of transcription, it is suggested here that isochores arose not as an adaptation to elevated temperature but because of a certain grade of general organization and correspondingly advanced level of genomic organization, reflected in genome structuring, with physical properties of DNA in the gene-rich regions being optimized for active transcription and in the gene-poor regions for chromatin condensation ('transcription/grade' concept).

Animals↗
PubMed2003

Shuffling of genes within low-copy repeats on 22q11 (LCR22) by Alu-mediated recombination events during evolution.

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der(22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5' or 3' ends of them. A possible stimulus for the 5' or 3' terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.

Alu Elements↗
PubMed1996

Sequence analysis of the conserved protamine gene cluster shows that it contains a fourth expressed gene.

Structural data are presented on the protamine gene cluster (PGC) of human, mouse, rat, and bull. By restriction mapping we demonstrate that the organization of the protamine cluster is conserved throughout all four species, i.e., the genes are situated in a head to tail arrangement in the order: protamine 1-protamine 2-transition protein 2. Further, we established the nucleotide sequence of the entire human PGC (25 kb in total) and the 3' portion of the rat protamine cluster (PRM2 and TNP2 genes and intergenic region). In addition, a 1 kb fragment of the bovine and murine protamine cluster, situated between PRM2 and TNP2, was sequenced. This fragment is conserved regarding sequence, position, and orientation in all species examined, and was classified as likely coding region by gene recognition program GRAIL. Using the rat fragment as a probe in RNA blots, we detected a testis-specific signal of about 0.5 kb. Finally, we demonstrate a high density of Alu elements, both full and fragmented copies, in the human PGC and discuss their localization with respect to evolutionary and functional aspects.

Animals↗
PubMed1993

Isolation and cloning by a polymerase chain reaction of a genomic DNA fragment of the human slow skeletal troponin (TNNT1) gene.

The genomic 3' structure of the gene coding for the human slow skeletal troponin T (TNNT1) gene, is reported. An intron of 912 nucleotides containing an Alu-element has been identified and characterized. The complexity of the sequenced region suggests an alternative exon use. The present results may be valuable for further studies on the gene structure of TNNT1 and the related troponin gene family.

Base Sequence↗
PubMed1992

The human immunoglobulin kappa locus. Characterization of the duplicated A regions.

The central regions of the kappa locus, the so-called A regions, have been fully characterized on cosmid and phage lambda clones. The regions, which are parts of the C kappa-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 V kappa genes and pseudogenes. The A regions have been linked on their 5' sides to the O regions and on their 3' sides to the L regions. Chromosomal walking has eliminated a previous gap in the Ap region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 V kappa genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5-kb fragment including one V kappa gene from Ap; the sequence divergence of duplicated V kappa gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the V kappa-J kappa rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the kappa locus.

Amino Acid Sequence↗
PubMed1991

Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination.

Three tandem duplications were previously identified in patients with Duchenne muscular dystrophy and were shown in each case to have a subset of dystrophin gene exons duplicated. The origin of these duplications was traced to the single X chromosome of the maternal grandfathers, suggesting that an intrachromosomal event (unequal sister chromatid exchange) was involved in the formation of these duplications. In the present study, a DNA segment containing the duplication junction and the normal DNA that corresponds to both ends of the duplicated region have been cloned. Subsequent mapping studies confirmed the tandem arrangement (head to tail) of these duplications and revealed their sizes to be 130 kb, approximately 300 kb, and 35-80 kb, respectively. Sequence analysis of the duplication junctions showed that one duplication was due to homologous recombination between two repetitive elements (Alu sequences) and the other two were due to recombination between unrelated nonhomologous sequences. In the latter cases, the preferred cleavage sites of the eukaryotic type I and II DNA topoisomerases were found at the junctions of these duplications, suggesting a possible role of these enzymes in the chromatid exchange events. This study provides the first insight into the molecular basis of gene duplications formed through unequal sister chromatid exchange in humans.

Base Sequence↗
PubMed1993

Splicing of an anti-sense Alu sequence generates a coding sequence variant for the alpha-3 subunit of a neuronal acetylcholine receptor.

In this report we demonstrate that an alpha-3 acetylcholine receptor subunit transcriptional variant originates through alternative splicing of a complementary sequence of the right arm of an Alu element. This element is located within the 5.1 Kb intron found between exons 5 and 6 of the alpha-3 acetylcholine receptor subunit gene. The transcriptional variant originates from the normal splicing process and carries an in-frame stop codon. If translated, it should encode for a peptide lacking the 4th transmembrane domain of the normal subunit.

Alternative Splicing↗
PubMed1994

Evolution of the chAB4 multisequence family in primates.

Approximately 50 members of the primate-specific multisequence family chAB4 are located as clusters at eight different chromosomal loci within the human genome. The whole cloned region of chAB4 represents a single-copy or low-copy sequence in all nonhuman primates tested, with the exception of the chimpanzee, for which we found chAB4 copy numbers similar to those in the human. An Alu element was inserted into chAB4 after the divergence of the Old World monkeys from the hominoids but before chAB4 was amplified. The first amplification step could be dated after the great apes and the human diverged from the Old World monkeys. We have evidence that neither the copy numbers nor the chromosomal locations remained stable after this initial step and that gross alterations in the relative copy numbers of individual family members occurred even after the divergence of the human and the chimpanzee. Taken together, our data suggest that chAB4, in an evolutionary sense, is an unusually unstable sequence family.

Animals↗
PubMed1997

Genomic structure and complete nucleotide sequence of the Batten disease gene, CLN3.

We recently cloned a cDNA for CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis or Batten disease. To resolve the genomic organization we used a cosmid clone containing CLN3 to sequence the entire gene in addition to 1.1 kb 5' of the start of the published CLN3 cDNA and 0.3 kb 3' to the polyadenylation site. CLN3 is organized into at least 15 exons spanning 15 kb and ranging from 47 to 356 bp. The 14 introns vary from 80 to 4227 bp, and all exon/intron junction sequences conform to the GT/AG rule. Numerous repetitive Alu elements are present within the introns and 5'- and 3'-untranslated regions. The 5' region of the CLN3 gene contains several potential transcription regulatory elements but no consensus TATA-1 box was identified. CLN3 is homologous to 27 deposited human ESTs, and sequence comparisons suggest alternative splicing of the gene and the existence of transcribed sequences upstream to the start of the published CLN3 cDNA.

Base Sequence↗
PubMed1997

Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD).

Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1998

The human prostate-specific transglutaminase gene (TGM4): genomic organization, tissue-specific expression, and promoter characterization.

Human prostate-specific transglutaminase (hTGP) is a cross-linking enzyme secreted by the prostate. In this study, we performed dot blot analysis of 50 normal human tissues to demonstrate unambiguously the prostate-specific expression of hTGP. Furthermore, we elucidated the genomic organization of the TGM4 gene, the gene encoding hTGP. The structure of this gene displays striking similarity to that of other transglutaminase (TGase) genes. The TGM4 gene spans approximately 35 kb of genomic DNA and consists of 13 exons and 12 introns. The main transcription initiation site is located 52 bp upstream of the translational start codon. A hTGP splice variant of intron 1 was detected. This splice variant contains an in-frame antisense Alu element insertion. The TGM4 promoter was analyzed by sequencing and transfection experiments. At positions -1276 to -563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA. Deletion mapping of the TGM4 promoter in the transiently transfected human prostate cancer cell line PC346C showed comparable activity of 2.1-, 1.5-, and 0.5-kb promoter fragments.

Amino Acid Sequence↗
PubMed1998

Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform.

Htra2-beta is a human homologue of Drosophila transformer-2 and a member of the SR-like protein family. Here we report the isolation and characterization of the complete htra2-beta gene (HGMW-approved symbol SFRS10). The gene spans 21,232 bp and is composed of 10 exons and 9 introns. Radiation hybrid mapping localized the gene to chromosome 3q. The region upstream of the transcription initiation codon contains an Alu element and several potential transcription factor binding sites. RT-PCR and comparison with EST clones revealed five different RNA isoforms generated by alternative splicing. These isoforms encode three diverging open reading frames, and two of these, htra2-beta3 and htra2-beta4, lack the first SR domain. Htra2-beta3 is developmentally regulated and expressed predominantly in brain, liver testis, and weakly in kidney. Furthermore, the domain structure of htra2-beta3 resembles a variant found in the Drosophila male germline, indicating a remarkable conservation of alternative transformer-2 variants. Finally, we show that htra2-beta3 is expressed in the nucleus and interacts with a subset of SR proteins in a yeast two-hybrid system and in vivo.

Alternative Splicing↗
PubMed2001

RET rearrangements in radiation-induced papillary thyroid carcinomas: high prevalence of topoisomerase I sites at breakpoints and microhomology-mediated end joining in ELE1 and RET chimeric genes.

Children exposed to radioactive iodine after the Chernobyl reactor accident frequently developed papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase gene. Various types of RET rearrangements have been described. More than 90% of PTC with RET rearrangement exhibit a PTC1 or PTC3 type of rearrangement with an inversion of the H4 or ELE1 gene, respectively, on chromosome 10. To obtain closer insight into the mechanisms underlying PTC3 inversions, we analyzed the genomic breakpoints of 22 reciprocal and 4 nonreciprocal ELE1 and RET rearrangements in 26 post-Chernobyl tumor samples. In contrast to previous assumptions, an accumulation of breakpoints at the two Alu elements in the ELE1 sequence was not observed. Instead, breakpoints are distributed in the affected introns of both genes without significant clustering. When compared to the corresponding wildtype sequences, the majority of breakpoints (92%) do not contain larger deletions or insertions. Most remarkably, at least one topoisomerase I site was found exactly at or in close vicinity to all breakpoints, indicating a potential role for this enzyme in the formation of DNA strand breaks and/or ELE1 and RET inversions. The presence of short regions of sequence homology (microhomologies) and short direct and inverted repeats at the majority of breakpoints furthermore indicates a nonhomologous DNA end-joining mechanism in the formation of chimeric ELE1/Ret and Ret/ELE1 genes.

Base Sequence↗
PubMed1999

Isolation and characterization of a novel transcript embedded within HIRA, a gene deleted in DiGeorge syndrome.

We have isolated a few cDNAs from different human tissues, transcribed from the first intron of HIRA, a gene deleted in the DiGeorge syndrome. These cDNAs are produced by an intronic gene (22k48) which is transcribed by the HIRA opposite strand and is itself arranged in exons and subjected to alternative splicing. The longest continuum cDNA sequence we obtained is 3.6 kb long and contains 3 different exons and 2 introns. 22k48 cDNA is composed of several tandemly arranged repeated elements (Alu, LINEs, CAn) surrounding a unique sequence. In situ hybridization showed the presence of 22k48 RNA in the cytoplasm of CNS and PNS neurons. 22k48 RNA is able to bind cytoplasmic proteins in the range of 45 to 60 kDa. 22k48 is a new member of the small group of genes that are transcribed but not translated, and its haploinsufficiency could contribute to the pathogenesis of the DiGeorge syndrome.

Adult↗
PubMed1993

Variable SINE 3' poly(A) sequences: an abundant class of genetic markers in the pig genome.

Previous studies on human DNA have shown that the 3' poly(A) tracts of Alu elements may display considerable genetic polymorphism. To explore whether this marker type is generally applicable in mammalian genomes, I analyzed porcine SINEs. A database screening revealed 17 porcine sequences with significant homology to a previously identified pig SINE. The occurrence in the database suggested a SINE frequency of one copy every 12 kb of pig DNA. All SINEs contained a 3' poly(A) tract with an average of 12 uninterrupted adenines. The repetitive regions were analyzed for polymorphism by locus-specific PCR amplification. Allelic length variation (two to five alleles among 10 pigs) was found at 8 out of 10 loci investigated, in most cases probably because of varying number of iterated adenine residues. There was a positive relationship between repeat length and the degree of polymorphism. Stable Mendelian inheritance was documented in 200 meioses each at four loci. The high genomic frequency of SINEs implies that a potentially informative marker may be found near any gene or in any cosmid clone. These SINE 3' poly(A) polymorphisms, termed SINEVA [SINE variable poly(A)s], thus provide an abundant and useful class of genetic marker in mammalian genomes.

Alleles↗
PubMed1993

Multiplication of Mhc-DRB5 loci in the orangutan: implications for the evolution of DRB haplotypes.

The beta chain-encoding (B) class II genes of the primate major histocompatibility complex belong to several families. The DRB family of class II genes is distinguished by the occurrence of haplotype polymorphism--the existence of multiple chromosomal forms differing in length, gene number, and gene combinations, each form occurring at an appreciable frequency in the population. Some of the haplotypes, or fragments thereof, are shared by humans, chimpanzees, and gorillas. In an effort to follow the DRB haplotype polymorphism further back in time, we constructed DRB contig maps of the two chromosomes present in the orangutan cell line CP81. Two types of genes were found in the two haplotypes, Popy-DRB5 and Popy-DRB1*03, the former occurring in two copies and one gene fragment in each haplotype, so that the CP81 cell line contains four complete DRB5 genes and two DRB5 fragments altogether. Since the four genes are more closely related to one another than they are to other DRB5 genes, they must have arisen from a single ancestral copy by multiple duplications. At the same time, however, the two CP81 haplotypes differ considerably in their restriction enzyme sites and in the presence of Alu elements at different positions, indicating that they have been separated for a length of time that exceeds the lifespan of a primate species. Moreover, a segment of about 100 kilobase pairs is shared between the orangutan CP81-1 and the human HLA-DR2 haplotype. These findings indicate that part of the haplotype polymorphism may have persisted for more than 13 million years, which is the estimated time of human-orangutan divergence.

Amino Acid Sequence↗
PubMed1992

Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses.

We have used a gamma-irradiation (2.5-25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14-150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27-28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3-30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2-3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1-2 Mb on average, a size range amenable to PFGE and YAC contig mapping.

Animals↗
PubMed1994

Compartmentalization of cellular and viral DNAs in adenovirus type 5 infection as revealed by ultrastructural in situ hybridization.

We describe the respective distributions of cellular DNA and viral genomes in adenovirus type 5 (Ad5)-infected HeLa cells by means of electron microscope in situ hybridization using biotinylated Alu DNA and Ad5 DNA probes in a post-embedding technique. When hybridization was performed on sections of formaldehyde-fixed material, Alu elements were restricted to the cellular chromatin, irrespective of the stage of the infectious cycle, without any interpenetration within the virus-induced regions. Viral DNA was confined entirely to the latter. Similar topological distributions of cellular and viral DNA were obtained when in situ hybridizations were performed at the optical level on methanol-acetone fixed cells. Such separation between cellular and viral DNA persisted under experimental conditions which partially loosened the nucleoproteins. In addition, the viral DNA of the different viral structures spread without interpenetration. On the other hand, the procedure did not dissociate the clusters of viruses which remained, as usual, closely surrounded by innumerable viral genomes. It also preserved the shapes, content (viral ssDNA + viral 72 kD protein) and the topological localization of the pleomorphic viral ssDNA accumulation sites. These data, which suggest that all viral DNAs are spatially linked in the infected nuclei, suggest a prominent role for the internal nuclear matrix in virus replication, assembly and maturation.

Adenoviruses, Human↗