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PubMed1984

The activation of cellular genes in transformed cells.

We have used differential cDNA cloning techniques to isolate a number of genes that are activated as a result of transformation by the DNA tumour virus Simian virus 40. From the nucleotide sequences of the cDNA clones we have been able to identify some of these genes. One of them derives from the major histocompatibility complex and contains a repetitive element that identifies a large number of RNAs present in pluripotential embryonic cells.

Animals↗
PubMed1997

A high-resolution physical and transcript map of the Cri du chat region of human chromosome 5p.

A high-resolution physical and transcription map has been generated of a 3.5-Mb region of 5p15.2 that is associated with the Cri du chat (CDC) syndrome. Utilizing a variety of resources including a natural deletion panel, a chromosome specific radiation hybrid panel, and YAC, PAC, and BAC genomic clones we have ordered > 60 STSs within this region. Approximately 45% of these STSs were obtained from publicly available whole genome maps, thus allowing for integration of this map with currently available resources. Thirteen of these markers were ESTs. In addition, > 70 exon trapped products have been mapped on the natural deletion panel and bacterial clone resource. The combination of these resources has allowed for the identification of 17 transcripts within this region, all of which represent candidate genes for CDC. Further characterization of the genomic contig also revealed that this region of 5p15 contains a large number of repetitive elements.

Base Sequence↗
PubMed1981

Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae.

CYC1 and sup4 are part of a tightly linked cluster of genes on chromosome X in the yeast Saccharomyces cerevisiae. Using as probes previously cloned fragments containing the CYC1 and sup4 genes, we have identified and cloned the deoxyribonucleic acid (DNA) present between these genes in one strain of yeast. We find that the CYC1 and sup4 genes are approximately 21 kilobases apart. In the same strain, the meiotic map distance is approximately 3.7 centimorgans, for a ratio of 5.6 kilobases per centimorgan in this interval. The physical mapping has allowed unambiguous determination of the orientation of CYC1 and sup4 relative to each other, the centromere, and a nearby transfer ribonucleic acid (tRNA(2Ser)) gene. The spontaneous mutation cyc1-1 inactivates the CYC1 gene as well as the neighboring loci OSM1 and RAD7. We have determined that a cyc1-1-bearing strain lacks approximately 13 kilobases of single-copy DNA from the CYC1-sup4 region, including all of the CYC1 coding information. There is a sequence homologous to the middle-repetitive element Ty1 at or near the breakpoint of the cyc1-1 deletion. We discuss the possibility that Ty elements play a role in the formation of such large, spontaneous deletions, which occur frequently in this region of chromosome X in certain yeast strains.

Chromosome Deletion↗
PubMed1996

A systematic survey of the intergenic region between the murine oxytocin- and vasopressin-encoding genes.

The genomic region between the oxytocin (OT) and vasopressin (VP) genes in the two strains of mice was independently sequenced by our two groups. In this report, we present our collated sequence data and analyses. The mouse intergenic region (MUIGR) was aligned to that of the rat, which has been reported to contain 6.4-kb long interspersed nuclear element (LINE). The MUIGR sequences in the two mice strains did not contain any LINE sequences. This suggests that the approximately 3.5-kb sequence that is conserved between the rat and mouse intergenic regions is likely to be involved in the regulation of OT and VP expression. We also observed several conserved putative transcription factor recognition sequences. Analysis of the MUIGR revealed the lack of any significant ORFs, but the presence of several repetitive elements.

Animals↗
PubMed1991

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.

Amino Acid Sequence↗
PubMed1991

Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs).

To facilitate studies of gene expression and homologous recombination, plasmids have been developed which permit the insertion of neomycin resistance-encoding gene (NmR) into either the human DNA insert or the vector arm of a yeast artificial chromosome (YAC). To integrate into the YAC arm, the plasmid pRV1 contains a LYS2 (encoding alpha-aminoadipate reductase) gene for selection in the yeast host, and a NmR gene for subsequent selection after transfection of mammalian cells. These two sequences are bracketed by fragments of the URA3 gene (encoding orotidine-5'-phosphate decarboxylase) that can disrupt the URA3 gene in the YAC arm by homologous recombination in yeast. To integrate a selectable marker into the insert, the plasmid pRV2 contains a NmR gene and an intact copy of the URA3 gene, bracketed by segments of an L1 (LINEs) repetitive element. In this case, the vector has been designed for use with YACs that have already been fitted in the vector arm with a different marker (i.e., TK) that has disrupted the URA3 gene in the vector arm. Selection is for the restoration of URA3 gene activity attendant on recombination into an L1 element in the YAC insert. Use of the vectors is illustrated with a YAC clone containing ribosomal DNA.

Aldehyde Oxidoreductases↗
PubMed2000

The skeletal muscle alpha-actin gene of channel catfish (Ictalurus punctatus) and its association with piscine specific SINE elements.

The alpha-actin gene of channel catfish (Ictalurus punctatus) was cloned and sequenced. The gene has a similar organization and exhibited a high level of sequence similarity to those from other vertebrate animals. The upstream region of the alpha-actin gene included a TATA box, a CAAT box, three E-boxes, and a CArG box. Nested deletion segments containing these transcriptional motifs were fused to the reporter gene chloramphenicol acetyl transferase (CAT). Transfection of the clones into C2C12 cells indicated that all these motifs are required for transcriptional activities. The channel catfish alpha-actin gene is associated with two distinct short interspersed repetitive elements (SINEs). The first SINE element showed high levels of sequence similarity to the zebrafish Mermaid element, while the second SINE element is not similar to the Mermaid element except for an 8bp sequence CCCCGTGC suggesting their evolutionary linkage. However, the second SINE element appeared to co-exist with the Mermaid element in most cases and therefore was designated as the Merman element. Approximately 9000 copies and 1200 copies of the Mermaid and Merman elements exist per haploid channel catfish genome, respectively. BLAST searches indicated that both the Mermaid and the Merman elements were frequently associated with gene sequences, mostly those of aquatic animals, suggesting their evolutionary origin in association with aquatic organisms and their function in shaping the evolution of genomes in aquatic animals.

Actins↗
PubMed1990

Molecular structures of two human DNA topoisomerase I retrosequences.

We have isolated recombinant lambda-phage clones that contain sequences complementary to the 3' half of the cDNA encoding human topoisomerase I (hTOP1). These lambda clones belong to three distinct classes: class-I clones contain sequences from the active gene located on human chromosome 20. Class-II and class-III clones contain sequences corresponding to the cDNA encoding hTOP1 from nucleotide (nt) 2208 to 3434 and from nt 1639 to 3434, respectively. These sequences exhibit the characteristic features of retroposons or retrosequences. They are most likely derived from truncated mRNA transcripts of the active gene. We propose to designate the truncated hTOP1 sequence located on chromosome 1 as the pseudogene 1 (psi 1-hTOP1) and the sequence on chromosome 22 as the pseudogene 2 (psi 2-hTOP1). Pseudogene psi 1-hTOP1 has two unique properties: it is flanked by upstream sequences which display promoter activity in transient expression assays, and it contains an open reading frame which could code for the 211 C-terminal amino acids of hTOP1. Pseudogene psi 2-hTOP1 is located within an AluI repetitive element and is flanked on one side by a (CA)21 stretch.

Base Sequence↗
PubMed2005

PEA3, AP-1, and a unique repetitive sequence all are involved in transcriptional regulation of the breast cancer-associated gene, mammaglobin.

The breast cancer-associated gene mammaglobin is a member of the secretoglobin protein family and has demonstrated its utility as a breast cancer marker. However, the transcriptional regulation of mammaglobin has not been well-characterized. In this report, we used luciferase reporter assays to identify the 200 bp directly 5' of the transcriptional start site as the minimal promoter region of mammaglobin. Sequence scanning indicated that two PEA3 transcription sites were possibly involved in mammaglobin transcription. By transfecting a PEA3 expression vector into breast cancer cell lines MDA-MB-415 and MCF-7, we determined that exogenous PEA3 was able to drive transcription. Mutational analysis indicated that each PEA3 site was functional. Our reporter system and electrophorectic mobility shift assays (EMSAs) also identified the involvement of a unique repetitive element in mammaglobin transcription. Finally, AP-1 was determined via luciferase assays to be involved in regulating non-PEA3 dependent transcription. Elucidating these cis-acting elements will impact our understanding of transcription of normal breast and breast cancer-associated genes.

Breast↗
PubMed1987

A systematic survey of repetitive sequences abundantly expressed in rat tumors.

A cDNA library made from poly(A)+ RNA of a rat ascites hepatoma cell line, AH60C, was screened with respect to transcribed repetitive sequences by probing with AH60C genomic DNA. Thirty clones, comprising 1.2% of the library, were found to contain repetitive elements. The sequences of 12 clones among them were abundantly expressed in poly(A)+ RNA of AH60C, yet sparse in that of rat normal liver. This differential expression was also noted in case of other tumors and normal tissues tested. These 12 clones could be divided into two classes, depending on whether their sequences were expressed in cytoplasmic poly(A)+ RNA (class I) or not (class II). Rat Alu-like type II (B2) repeats and an unknown repetitive sequence were found in the clones of class I. Adjacent to each of them, we detected two non-repeated sequences, the expressions of which were also activated. The presence of another unknown repetitive sequence was observed in the clones of class II. It is noted that more than one-third of the transcribed repetitive sequences were involved in the predominant expression in tumor cells, whereas the majority of overall transcripts have been found to be common to both transformed cells and their normal counterparts.

Animals↗
PubMed1985

Activation of putative transposition intermediate formation in tumor cells.

The transcription levels of two families of mouse repetitive elements namely intracisternal A particle (IAP) genes, and B2 sequences were analyzed in different tumor cells and normal tissues. These sequences belong to two major classes of mobile elements present in the mouse genome. The Northern blots containing poly(A) + RNAs from tumor cells and normal tissues were hybridized to the cloned IAP gene and B2 sequence. The content of IAP gene transcripts in tumor cells is much higher than in normal cells. A 10- to 100-fold difference was found. The predominant IAP-gene specific RNAs in all investigated tumor cells were 9.5, 6.8 and 5.3 kb long. Additional RNA species were found in some of the tumors. The active synthesis of small cytoplasmic B2 RNA transcribed by RNA polymerase III was also detected in most tumor cells tested. Usually it was higher than in normal cells. Free closed circular DNAs hybridizing to IAP gene probes were cloned from Ehrlich ascites carcinoma cells. We speculate that the data obtained indicate the enhanced transposition of mobile elements in tumor cells which may be an important factor of tumor progression.

Animals↗
PubMed1985

opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster.

The principal transcription product of Notch, a locus involved in the neurogenesis of D. melanogaster, is a developmentally regulated poly(A)+ RNA approximately 10.5 kb in length. Analysis of the structure of this RNA has revealed a 93 bp repeated sequence that is shared by many other developmentally regulated transcription units. Nucleotide sequence analysis of the repeat shows an unusual structure consisting predominantly of the triplets CAG and CAA, both of which can code for the amino acid Gln. We present evidence indicating that the Notch repeat is a member of a novel family of repetitive elements, which we term the opa family. Our data suggest that some of these elements may be not only transcribed but also translated. We compare opa with other known transcribed repeats and speculate on its functional significance.

Amino Acid Sequence↗
PubMed1995

A promoter associated with the neisserial repeat can be used to transcribe the uvrB gene from Neisseria gonorrhoeae.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrB mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrB proteins of E. coli, Micrococcus luteus, and Streptococcus pneumoniae. A gonococcal uvrB mutant was constructed and found to be extremely sensitive to UV radiation. Transcriptional fusions between portions of the gonococcal uvrB upstream region and a reporter gene were used to localize promoter activity, and the transcriptional start point of the gonococcal uvrB gene was mapped in E. coli by primer extension. A corresponding sigma 70 promoter was identified within a copy of the 26-bp neisserial repeat, and this identification provided the first evidence of a promoter associated with this repetitive element in N. gonorrhoeae.

Amino Acid Sequence↗
PubMed1998

Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome.

The pattern of histone H4 acetylation in different genomic regions has been investigated by immunoprecipitating oligonucleosomes from a human lymphoblastoid cell line with antibodies to H4 acetylated at lysines 5, 8, 12 or 16. DNA from antibody-bound or unbound chromatin was assayed by slot blotting. Pol I and pol II transcribed genes located in euchromatin were shown to have levels of H4 acetylation at lysines 5, 8 and 12 equivalent to those in input chromatin, but to be slightly enriched in H4 acetylated at lysine 16. In no case did the acetylation level correlate with actual or potential transcriptional activity. All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromatin and with the isoform. Two single copy genes that map within or adjacent to blocks of paracentric heterochromatin are depleted in H4 acetylated at lysines 5, 8 and 12, but not 16. Consensus sequences of repetitive elements of the Alu family (SINES, enriched in R bands) were associated with H4 that was more highly acetylated at all four lysines than input chromatin, while H4 associated with Kpn I elements (LINES, enriched in G bands) was significantly underacetylated.

Acetylation↗
PubMed2000

Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus.

The platyfish (Xiphophorus maculatus), in which sex chromosomes are evident from stable and predictable inheritance of sex, is one of the best-studied lower vertebrates with respect to sex determination. In order to identify the structural equivalent for this in the karyotype, which does not contain heteromorphic pairs of chromosomes, two sex-linked molecular probes were used for fluorescent in situ hybridization analysis. One probe, derived from the melanoma oncogene locus ONC-Xmrk, stained both the X and the Y chromosome. This cytogenetic analysis mapped the sex-determining locus to the subtelomeric region of a medium-sized telocentric chromosome. Another probe, a repetitive element (XIR), specifically labeled the Y chromosome in metaphase spreads and in interphase nuclei. The sex chromosomes of X. maculatus can be considered to be at an early stage of evolution of gonosomes. Expansion of the XIR repeat is obviously one of the earliest of the molecular events that lead to divergence of the Y chromosome and recombinational isolation of the sex-determining locus.

Alleles↗
PubMed2005

Comparative genomics of Dictyostelium discoideum and Entamoeba histolytica.

Amoebozoa represent one of the earliest branches from the last common ancestor of all eukaryotes and contain some of the most dangerous human pathogens. Two amoebozoan genomes -- from the model organism Dictyostelium discoideum and the human pathogen Entamoeba histolytica -- have been published this year. Owing to their high A+T content, both genomes were difficult to sequence. In addition to nine amoebozoan expressed sequence tag projects, efforts are underway for comparative sequencing of four additional Entamoeba species. The completed genome sequences of D. discoideum and E. histolytica revealed unusual telomere structures, a high percentage of repetitive elements and a remarkably high gene content that is close to the one of Drosophila melanogaster. Finally, both organisms are brilliant examples of the influence of the lifestyle of an organism on its genome.

Adaptation, Biological↗
PubMed1995

Isolation and characterization of a retroelement from B chromosome (PSR) in the parasitic wasp Nasonia vitripennis.

Molecular characterization of the paternal-sex-ratio (PSR) chromosome in Nasonia vitripennis (Hymenoptera: Pteromalidae) has led to the isolation of a dispersed repetitive element. The element is a LTR-containing retrotransposon which has been named NATE (NAsonia Transposable Element). NATE has direct terminal repeats and has an internal amino acid sequence similar to reverse transcriptases of other retroelements. Phylogenetic analysis indicates NATE is a member of the Gypsy/Ty3 group of retrotransposons, and represents the first isolated from Hymenoptera. Five closely related copies of NATE were isolated from the PSR chromosome, but cross-hybridizing elements were not detected on the autosomes of N. vitripennis. Strongly cross-hybridizing elements were, however, detected in two other Nasonia species. This observed distribution of NATE is interesting, because the supernumerary PSR chromosome may be derived from the genome of a sibling species of N. vitripennis.

Amino Acid Sequence↗
PubMed1996

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped.

Amino Acid Sequence↗