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At least 1,135 records · Page 63Linked to original sources
PubMed2000

Genomic structure of the human p47-phox (NCF1) gene.

The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system. Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen. A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD). We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals. The gene is 15,236 base pairs long and includes 11 exons. It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23. Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements. In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns. These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease. Analysis of 1.96 kb of sequence 5' of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones. Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals.

5' Untranslated Regions↗
PubMed1983

Human immune interferon (IFN-gamma) gene sequence and structure.

Human IFN-gamma is encoded by a single gene which, in contrast to the IFN-alpha and IFN-beta genes, contains three introns. This gene has been isolated from a recombinant human-gamma gene library, and the entire gene and 5' and 3' flanking regions have been sequenced. Southern hybridizations failed to show any polymorphism of the gene or its flanking regions. The first intron contains a repetitive element which is unrelated to the Alu family repeat.

Amino Acid Sequence↗
PubMed1996

A human B-box-binding protein downregulated in adenovirus 5-transformed human cells.

Internal promoters of some genes transcribed by RNA polymerase III (e.g. tRNA genes, adenovirus VA1 RNA gene, human retroposons of the Alu family) contain a conserved sequence element, B-box, interacting with basal transcription factor TFIIIC2 which initiates assembly of the full transcription complex on the genes, and which represents the major determinant of the efficiency of their expression. In this study we have identified in human nuclear extracts a protein which interacts with VA1 B-box DNA and forms a high-affinity complex which is very stable after the addition of a large excess of competitor DNA. Unlike TFIIIC2, the B-box-binding activity of the B-box-binding protein is found to be decreased in adenovirus 5-transformed human cells. In these cells (line 293) increased transcription of VA1 and tRNA genes in vivo and in vitro was previously detected by other workers. Our results suggest that besides TFIIIC2, an additional B-box-binding protein factor may be involved in the regulation of expression of the RNA polymerase III-transcribed genes.

Adenoviruses, Human↗
PubMed1996

Molecular cloning of the murine branched chain alpha-ketoacid dehydrogenase E2 subunit: presence of 3' B1 repeat elements.

The cDNA sequence encoding the murine E2 subunit (dihydrolipoyl transacylase) of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex was determined. In the region encoding the mature E2 subunit protein, both the nucleotide composition and predicted amino acid sequence are highly conserved between murine, human, and bovine species. In contrast, the 5' sequence encoding the amino-terminal preprotein sequence and 3' untranslated region are less well conserved. The 3'-noncoding region contains sequences highly homologous to the rodent B1 repeat elements, which are related to human Alu repeat sequences. This finding is similar to the presence of three Alu repeat sequences in the 3'-noncoding region of human E2 cDNA.

Acyltransferases↗
PubMed1995

Regulation of the human poly(ADP-ribosyl) transferase promoter via alternative DNA racket structures.

Human nuclear poly(ADP-ribosyl) transferase (ADPRT) protein content in cells suggests that ADPRT expression is stringently controlled. Analysis of the 3 kb promoter sequence, which is required for high level expression, revealed an extraordinary architecture: several Sp1 motifs are located in the vicinity of the first exon but the closest CCAAT/TATA boxes are several hundred basepairs away. Four Alu type repetitive sequences are in the promoter structure. Within these Alu sequences there exist inverted repeat elements, which could form two mutually exclusive types of DNA tertiary structure consisting of quadruplex DNA and loops resembling rackets. Thereby, a CCAAT/TATA element would be moved to spatial vicinity of the Sp1 site activating the promoter. Deletion analysis showed the functional significance of these racket elements. We also obtained evidence for DNA racket structures when we studied mutational mechanisms in a human adenine phosphoribosyltransferase (APRT) deficient patient. One of his alleles harbours a novel complex type of deletion/insertion mutation. Based on several highly informative sequence features in this genomic region a model is proposed for the generation of this unusual type of mutation involving two steps: an initial targeting step and a subsequent complex rearrangement. This process includes the formation of a DNA racket structure, which resembles that of the ADPRT promoter. Thus we conclude that DNA racket structures seem to be of general importance in nature.

Base Sequence↗
PubMed1993

Measurement of recombination frequencies between two homologous DNA segments embedded in a YAC vector.

We measured the frequencies of recombination in a yeast host between two homologous segments of DNA that had been inserted with the same polarity in a yeast artificial chromosome (YAC) vector. Three kinds of YAC clones were constructed in which the gene encoding neomycin(Nm) resistance was sandwiched between two homologous segments of DNA, such as the IS3 elements of Escherichia coli or human Alu sequences. Frequencies of homologous recombination in yeast were measured in terms of loss of resistance to Nm. In the case of IS3 fragments, homologous recombination between them did occur at a relatively high frequency (5 x 10(-4). In contrast, recombination between two Alu sequences did not occur at a detectable level during a 30-day incubation. Thus, the frequency was less than 10(-5). These results indicate that the Alu sequences do not sufficiently promote the frequency of recombination between two homologous fragments in yeast as to induce rearrangements of DNA in a substantial fraction of YAC clones in libraries.

Base Sequence↗
PubMed1993

Structural organization of the human cardiac alpha-myosin heavy chain gene (MYH6).

The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, alpha and beta, that exist in tandem array on chromosome 14q12. We have previously sequenced the entire human cardiac beta-MyHC gene and now report the complete nucleotide sequence of the human cardiac alpha-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translation initiation codon. With the exception of the 13th intron of the human cardiac beta-MyHC, which is not present within the alpha-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the alpha-MyHC gene include four Alu repeats, a single (GT)n element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human alpha-MyHC cDNA sequence reveals several potential polymorphisms.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence and structure of the human apolipoprotein E gene.

The gene for human apolipoprotein E (apo-E) was selected from a library of cloned genomic DNA by screening with a specific cDNA hybridization probe, and its structure was characterized. The complete nucleotide sequence of the gene as well as 856 nucleotides of the 5' flanking region and 629 nucleotides of the 3' flanking region were determined. Analysis of the sequence showed that the mRNA-encoding region of the apo-E gene consists of four exons separated by three introns. In comparison to the structure of the mRNA, the introns are located in the 5' noncoding region, in the codon for glycine at position -4 of the signal peptide region, and in the codon for arginine at position +61 of the mature protein. The overall lengths of the apo-E gene and its corresponding mRNA are 3597 and 1163 nucleotides, respectively; a mature plasma protein of 299 amino acids is produced by this gene. Examination of the 5' terminus of the gene by S1 nuclease mapping shows apparent multiple transcription initiation sites. The proximal 5' flanking region contains a "TATA box" element as well as two nearby inverted repeat elements. In addition, there are four Alu family sequences associated with the apo-E gene: an Alu sequence located near each end of the gene and two Alu sequences located in the second intron. This knowledge of the structure permits a molecular approach to characterizing the regulation of the apo-E gene.

Amino Acid Sequence↗
PubMed1994

OXBOX and REBOX, overlapping promoter elements of the mitochondrial F0F1-ATP synthase beta subunit gene. OXBOX/REBOX in the ATPsyn beta promoter.

Three positive (PR1-3) and one negative (NR1) transcriptional control domain have been tentatively mapped in the promoter of the human F0F1-ATP synthase beta subunit gene (ATPsyn beta) in the context of expression in myogenic cells. Lipofection of promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells revealed that two of the three positive domains (PR1 and PR2) function in both myoblasts and myotubes, whereas the third positive domain (PR3) and the sole negative domain (NR1) seem to function only in myotubes. PR1 contains a cluster of four CCAAT cis-elements, PR2 is a small 44-base pair region containing an SP1-like motif, and PR3 is a region previously shown to be recognized by both OXBOX- and REBOX-binding factors. By site-directed polymerase chain reaction linker mutations, the activity of the OXBOX/REBOX cis-element in myoblasts is shown to be masked by flanking sequences in PR3. The negative domain, NR1, is located between 300 and 1,000 base pairs upstream from the OXBOX/REBOX elements in a region containing multiple Alu repeats. Mobility gel shift analysis of DNA-protein complexes using competitor DNAs verified the involvement of both OXBOX- and REBOX-binding factors in PR3. Similar experiments show SP1-specific binding at PR2. These data with observations of OXBOX and REBOX-specific binding of an OXBOX/REBOX-like region within the conserved sequence block C of the human mitochondrial DNA D-loop sequence are consistent with the idea that OXBOX- and REBOX DNA-binding factors coordinate the expression of mitochondrial energy genes in highly oxidative tissues by working with well characterized general transcription factors such as SP1 and CCAAT DNA-binding proteins, which exist in the nucleus, and MTF, which exists in the mitochondrion.

Animals↗
PubMed1991

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Whole-genome screening indicates a possible burst of formation of processed pseudogenes and Alu repeats by particular L1 subfamilies in ancestral primates.

BACKGROUND: Abundant pseudogenes are a feature of mammalian genomes. Processed pseudogenes (PPs) are reverse transcribed from mRNAs. Recent molecular biological studies show that mammalian long interspersed element 1 (L1)-encoded proteins may have been involved in PP reverse transcription. Here, we present the first comprehensive analysis of human PPs using all known human genes as queries. RESULTS: The human genome was queried and 3,664 candidate PPs were identified. The most abundant were copies of genes encoding keratin 18, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein L21. A simple method was developed to estimate the level of nucleotide substitutions (and therefore the age) of PPs. A Poisson-like age distribution was obtained with a mean age close to that of the Alu repeats, the predominant human short interspersed elements. These data suggest a nearly simultaneous burst of PP and Alu formation in the genomes of ancestral primates. The peak period of amplification of these two distinct retrotransposons was estimated to be 40-50 million years ago. Concordant amplification of certain L1 subfamilies with PPs and Alus was observed. CONCLUSIONS: We suggest that a burst of formation of PPs and Alus occurred in the genome of ancestral primates. One possible mechanism is that proteins encoded by members of particular L1 subfamilies acquired an enhanced ability to recognize cytosolic RNAs in trans.

Algorithms↗
PubMed2006

dbRIP: a highly integrated database of retrotransposon insertion polymorphisms in humans.

Retrotransposons constitute over 40% of the human genome and play important roles in the evolution of the genome. Since certain types of retrotransposons, particularly members of the Alu, L1, and SVA families, are still active, their recent and ongoing propagation generates a unique and important class of human genomic diversity/polymorphism (for the presence and absence of an insertion) with some elements known to cause genetic diseases. So far, over 2,300, 500, and 80 Alu, L1, and SVA insertions, respectively, have been reported to be polymorphic and many more are yet to be discovered. We present here the Database of Retrotransposon Insertion Polymorphisms (dbRIP; http://falcon.roswellpark.org:9090), a highly integrated and interactive database of human retrotransposon insertion polymorphisms (RIPs). dbRIP currently contains a nonredundant list of 1,625, 407, and 63 polymorphic Alu, L1, and SVA elements, respectively, or a total of 2,095 RIPs. In dbRIP, we deploy the utilities and annotated data of the genome browser developed at the University of California at Santa Cruz (UCSC) for user-friendly queries and integrative browsing of RIPs along with all other genome annotation information. Users can query the database by a variety of means and have access to the detailed information related to a RIP, including detailed insertion sequences and genotype data. dbRIP represents the first database providing comprehensive, integrative, and interactive compilation of RIP data, and it will be a useful resource for researchers working in the area of human genetics.

Databases, Genetic↗
PubMed2006

The evolution of mobile DNAs: when will transposons create phylogenies that look as if there is a master gene?

Some families of mammalian interspersed repetitive DNA, such as the Alu SINE sequence, appear to have evolved by the serial replacement of one active sequence with another, consistent with there being a single source of transposition: the "master gene." Alternative models, in which multiple source sequences are simultaneously active, have been called "transposon models." Transposon models differ in the proportion of elements that are active and in whether inactivation occurs at the moment of transposition or later. Here we examine the predictions of various types of transposon model regarding the patterns of sequence variation expected at an equilibrium between transposition, inactivation, and deletion. Under the master gene model, all bifurcations in the true tree of elements occur in a single lineage. We show that this property will also hold approximately for transposon models in which most elements are inactive and where at least some of the inactivation events occur after transposition. Such tree shapes are therefore not conclusive evidence for a single source of transposition.

Alu Elements↗
PubMed1991

Binding specificity of human nuclear protein interacting with the Alu-family DNA repeats.

We have demonstrated earlier that human cells contain nuclear protein interacting with conserved GC-rich sequence motifs of human Alu-family DNA repeats. One of these sequences is located in the region between elements A and B of bipartite RNA polymerase III promoter of Alu (AB-region). In this study we have used a DNase I footprinting assay with an Alu restriction subfragment covering AB-region, as well as a gel mobility shift assay with appropriate synthetic oligonucleotides to analyse in more detail the interaction of the protein with AB-region. We have also used antibodies raised against a zinc-finger peptide to examine the presence of a zinc-finger in the Alu-binding protein. The results indicate that AGG triplets may be important for high-affinity binding of the protein to DNA, and that the Alu-binding protein is a zinc-finger protein.

Amino Acid Sequence↗
PubMed2005

Polymorphic Alu insertions within the Major Histocompatibility Complex class I genomic region: a brief review.

Most polymorphic Alu insertions (POALINs) belong to a subgroup of the Alu multicopy retrotransposon family of short interspersed nucleotide elements (SINEs) that are categorized as AluYb8 and AluYa5. The number of AluYb8/AluYa5 members (approximately 4,492 copies) is significantly less than the approximately one million fixed Alu copies per human genome. We have studied the presence of POALINs within the Major Histocompatibility Complex (MHC) class I region on the short arm of chromosome 6 (6p21.3) because this region has a high gene density, many genes with immune system functions, large sequence variations and diversity, duplications and redundancy, and a strong association with more than 100 different diseases. Since little is known about POALINs within the MHC genomic region, we undertook to identify some of the members of the AluYb8/AluYa5 subfamily and to study their frequency of distribution and genetic characteristics in different populations. As a result of our comparative genomic analyses, we identified the insertion sites for five POALINs distributed within the MHC class I region. This brief review outlines the locations of the insertions and sequence features of the five MHC POALINs, their single site and haplotype frequencies in different geographic populations, and their association with different HLA class I genes and disease. We show that the MHC POALINs have a potential value as lineage and linkage markers for the study of human population genetics, disease associations, genomic diversity and evolution.

Chromosome Mapping↗
PubMed1991

Modulation of HeLa cell growth by transfected 7SL RNA and Alu gene sequences.

Alu and 7SL RNA gene sequences were tested for the potential to regulate mammalian cell growth by introducing these sequences into HeLa cells in a coupled DEAE-dextran transfection/affinity cell sorting system. Both Alu and 7SL RNA genes mediated inhibition of [3H]thymidine and [35S]methionine incorporation in recipient cells. In addition, short term growth curves were performed on calcium phosphate/DNA cotransfected, affinity-purified cells. This second assay revealed that transfected Alu and 7SL RNA can also cause suppression of HeLa cell proliferation. To investigate whether transcription or polymerase III (pol III) transcription factor binding was required for inhibitory activity, mutations were introduced into RNA pol III B block promoter elements in each of these genes. Suppression of [3H]thymidine incorporation was dependent on the presence of this pol III element in both genes; likewise, 7SL RNA-mediated growth suppression required the presence of the pol III B block promoter element. The results of this study indicate that Alu and 7SL RNA gene sequences interact with cellular factors that are important for HeLa cell proliferation and suggest that these pol III-transcribed elements may be involved in the regulation of cellular growth.

Base Sequence↗
PubMed1990

Characterization of the Alu-rich 5'-flanking region of the human prothrombin-encoding gene: identification of a positive cis-acting element that regulates liver-specific expression.

The nt sequence of 6127 bp of sequence upstream of the human prothrombin-encoding gene (F2) has been determined. Since we previously characterized 417 bp of DNA immediately upstream from the transcription start point (tsp), 6544 bp of continuous flanking sequence are known. Eleven Alu repeat sequences present in this region comprise 45% of the sequence; other repetitive sequences were identified by searching GenBank. The tsp was found to be heterogeneous by exon mapping and primer extension analysis. To localize the cis-acting sequences responsible for the liver-specific expression of F2, hybrid cat genes were constructed with various lengths of F2 5'-flanking region cloned upstream from a promoterless cat gene. After transfection into HepG2 and HeLa cells, it was inferred that the region between nt -1101 and -798 was required for synthesis in HepG2 cells; no synthesis was observed using these constructs in HeLa cells. Two sequences for known liver-specific or regulatory cis-acting sequences were identified in this region.

Amino Acid Sequence↗
PubMed1996

Mosaic evolution of rodent B1 elements.

We have determined sequences of PCR-amplified B1 elements from hamster and rat (Myomorpha), chipmunk (Sciuromorpha), and guinea pig (Caviomorpha). Between three and six B1 subfamilies were found in these species. In the phylogenetic analysis B1 sequences of hamster, mouse, and rat clustered separately from those of chipmunk and those of guinea pig. This is consistent with an independent evolution of B1 elements in separate rodent lineages. We exclude the possibility of convergent mutations to explain certain diagnostic characters within the modern B1 quasi-dimers and view these elements as mosaic structures assembling preexisting mutations. Furthermore, the presence of Alu-like structural motifs supports the hypothesis of the monophyletic origin of Alu and B1 repeats, i.e., from a common 7SL RNA-derived retroposing monomeric element.

Animals↗