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PubMed1997

CCAAT displacement protein (CDP/cut) recognizes a silencer element within the lactoferrin gene promoter.

Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non-LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing base-pair (bp) -387 to bp -726 of the LF promoter, but not in a -916-bp plasmid. Transfection of a -916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the -916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non-LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non-LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor-induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

Base Sequence↗

PubMed1998

Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction.

Anopheles arabiensis, one of the two most potent malaria vectors of the gambiae complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion 2Rd' of A. arabiensis. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different A. arabiensis strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element Odysseus. The presence of Odysseus at the junction of the naturally occuring inversion 2Rd' suggests that the inversion may be the result of the transposable element's activity. Characteristics of Odysseus' terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of Odysseus.

Animals↗

PubMed1987

Isolation and characterization of a nematode transposable element from Panagrellus redivivus.

We have isolated a transposable element, designated PAT-1, from the free-living nematode Panagrellus redivivus. P. redivivus strain C15 was found to have a high spontaneous mutation frequency compared to the standard Caenorhabditis elegans laboratory strain N2. To characterize the genetic lesions occurring in spontaneous C15 mutants, we molecularly cloned the homolog of the C. elegans unc-22 gene from wild-type P. redivivus and two strains carrying spontaneous mutations in this gene. One of these mutations resulted from the insertion of a 4.8-kilobase segment of repetitive DNA. This repetitive element (PAT-1) varies in copy number (10-50 copies) and location in different P. redivivus strains and is absent from C. elegans. The element could be useful as a transformation vector for C. elegans. Our approach is a general one that could be used to isolate additional nematode transposons from other species.

Animals↗

PubMed1998

Primer design for large scale sequencing.

We have developed PRIDE, a primer design program that automatically designs primers in single contigs or whole sequencing projects to extend the already known sequence and to double strand single-stranded regions. The program is fully integrated into the Staden package (GAP4) and accessible with a graphical user interface. PRIDE uses a fuzzy logic-based system to calculate primer qualities. The computational performance of PRIDE is enhanced by using suffix trees to store the huge amount of data being produced. A test set of 110 sequencing primers and 11 PCR primer pairs has been designed on genomic templates, cDNAs and sequences containing repetitive elements to analyze PRIDE's success rate. The high performance of PRIDE, combined with its minimal requirement of user interaction and its fast algorithm, make this program useful for the large scale design of primers, especially in large sequencing projects.

Algorithms↗

PubMed2006

Linkage disequilibrium of evolutionarily conserved regions in the human genome.

BACKGROUND: The strong linkage disequilibrium (LD) recently found in genic or exonic regions of the human genome demonstrated that LD can be increased by evolutionary mechanisms that select for functionally important loci. This suggests that LD might be stronger in regions conserved among species than in non-conserved regions, since regions exposed to natural selection tend to be conserved. To assess this hypothesis, we used genome-wide polymorphism data from the HapMap project and investigated LD within DNA sequences conserved between the human and mouse genomes. RESULTS: Unexpectedly, we observed that LD was significantly weaker in conserved regions than in non-conserved regions. To investigate why, we examined sequence features that may distort the relationship between LD and conserved regions. We found that interspersed repeats, and not other sequence features, were associated with the weak LD tendency in conserved regions. To appropriately understand the relationship between LD and conserved regions, we removed the effect of repetitive elements and found that the high degree of sequence conservation was strongly associated with strong LD in coding regions but not with that in non-coding regions. CONCLUSION: Our work demonstrates that the degree of sequence conservation does not simply increase LD as predicted by the hypothesis. Rather, it implies that purifying selection changes the polymorphic patterns of coding sequences but has little influence on the patterns of functional units such as regulatory elements present in non-coding regions, since the former are generally restricted by the constraint of maintaining a functional protein product across multiple exons while the latter may exist more as individually isolated units.

Biological Evolution↗

PubMed1992

Molecular and genomic organization of clusters of repetitive DNA sequences in Caenorhabditis elegans.

Repetitive sequences in Caenorhabditis elegans are interspersed along the holocentric chromosomes. We have physically mapped some of these repetitive families and found that, although the distribution of members of each family is relatively even along the chromosomes, members of more than one family tend to cluster in some locations. We compared the sequence organization of 11 clusters located at known positions on different chromosomes in the N2 strain. These studies allow a comparison between repetitive elements belonging to the same family that are located on the same or on different chromosomes, providing an important tool in the study of genome turnover and evolution.

Animals↗

PubMed1987

Analysis of a human gene cluster coding for tRNA(GAAPhe) and tRNA(UUULys).

A 13.8-kb fragment of human DNA isolated from a human lambda Charon-4A DNA library was found to contain four human tRNA genes. Nucleotide sequence analysis of approx. 3.7 kb of this segment of human DNA identified two lysine tRNA(UUU) genes identical in coding sequence to a previously reported human lysine tRNA gene [Roy et al., Nucl. Acids Res. 10 (1982) 7313-7322]. The other two tRNA genes were phenylalanine tRNA(GAA) genes, the first to be isolated from a mammalian source. These phenylalanine tRNA(GAA) genes were identical in sequence with the exception of a G/A polymorphism at coordinate 57. None of these tRNA genes contains introns. The tRNA(UUULys) and tRNA(GAAPhe) genes are organized in alternating order and are irregularly spaced, by intergenic regions of approx. 1.0, 2.6 and 5.0 kb, and randomly oriented. There was no evidence to indicate that any of these genes arose by gene duplication, since flanking sequence homology was limited to the putative RNA polymerase III termination signals in the 3'-flanking regions. A mature tRNA-sized product was identified following the transcription of each tRNA gene in a homologous in vitro transcription system. Interestingly, different levels of transcriptional activity of the three identical lysine tRNA genes were observed, suggesting modulation of tDNA expression by extragenic sequences. In addition, a minimum of eight regions of homology to Alu-type repetitive elements were detected in this human DNA fragment, one of which was located 53 bp upstream from a tRNA(GAAPhe) gene.

Base Sequence↗

PubMed1990

A cloned AluI-equivalent repetitive sequence that detects transfected hamster DNA.

An AluI-equivalent interspersed repetitive element showing 75% homology to a published consensus sequence, which is present at a level of 3 x 10(5) copies per hamster genome [Haynes et al., Mol. Cell. Biol. 1 (1981) 573-583], has been cloned into plasmid pUC8 as a tandem copy which can be readily excised, self-ligated and labelled for use as a hybridisation probe. This probe successfully detects integrated hamster sequences in the DNA of transfected human cells [Arrand et al., Proc. Natl. Acad. Sci. USA 86 (1989) 6997-7001], whereas repetitive hamster DNA isolated as a C0t = 1 (fast reannealing) fraction fails to do so. Hamster DNA-containing clones in genomic libraries derived from hamster/human hybrids are also easily detected with this probe, thus facilitating the isolation of hamster genes which complement human gene defects.

Animals↗

PubMed2001

Evolutionary analyses of the human genome.

The completion of the human genome will greatly accelerate the development of a new branch of science--evolutionary genomics. We can now directly address important questions about the evolutionary history of human genes and their regulatory sequences. Computational analyses of the human genome will reveal the number of genes and repetitive elements, the extent of gene duplication and compositional heterogeneity in the human genome, and the extent of domain shuffling and domain sharing among proteins. Here we present some first glimpses of these features.

Conserved Sequence↗

PubMed1997

Concerted evolution at a multicopy locus in the protozoan parasite Theileria parva: extreme divergence of potential protein-coding sequences.

Concerted evolution of multicopy gene families in vertebrates is recognized as an important force in the generation of biological novelty but has not been documented for the multicopy genes of protozoa. A multicopy locus, Tpr, which consists of tandemly arrayed open reading frames (ORFs) containing several repeated elements has been described for Theileria parva. Herein we show that probes derived from the 5'/N-terminal ends of ORFs in the genomic DNAs of T. parva Uganda (1,108 codons) and Boleni (699 codons) hybridized with multicopy sequences in homologous DNA but did not detect similar sequences in the DNA of 14 heterologous T. parva stocks and clones. The probe sequences were, however, protein coding according to predictive algorithms and codon usage. The 3'/C-terminal ends of the Uganda and Boleni ORFs exhibited 75% similarity and identity, respectively, to the previously identified Tpr1 and Tpr2 repetitive elements of T. parva Muguga. Tpr1-homologous sequences were detected in two additional species of Theileria. Eight different Tpr1-homologous transcripts were present in piroplasm mRNA from a single T. parva Muguga-infected animal. The Tpr1 and Tpr2 amino acid sequences contained six predicted membrane-associated segments. The ratio of synonymous to nonsynonymous substitutions indicates that Tpr1 evolves like protein-encoding DNA. The previously determined nucleotide sequence of the gene encoding the p67 antigen is completely identical in T. parva Muguga, Boleni, and Uganda, including the third base in codons. The data suggest that concerted evolution can lead to the radical divergence of coding sequences and that this can be a mechanism for the generation of novel genes.

Amino Acid Sequence↗

PubMed2000

UTRdb and UTRsite: specialized databases of sequences and functional elements of 5' and 3' untranslated regions of eukaryotic mRNAs.

The 5' and 3' untranslated regions of eukaryotic mRNAs may play a crucial role in the regulation of gene expression controlling mRNA localization, stability and translational efficiency. For this reason we developed UTRdb, a specialized database of 5' and 3' untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases including the presence of nucleotide sequence patterns already demonstrated by experimental analysis to have some functional role. All these patterns have been collected in the UTRsite database so that it is possible to search any input sequence for the presence of annotated functional motifs. Furthermore, UTRdb entries have been annotated for the presence of repetitive elements. All internet resources implemented for retrieval and functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs are accessible at http://bigarea.area.ba.cnr.it:8000/EmbIT/UTRH ome/

3' Untranslated Regions↗

PubMed1994

Organization of the human genome.

The human genome contains about 3 x 10(9) base pairs which, based on average estimates for the size of coding regions, might suggest an upper limit of about 3 million genes. Evidence from a variety of approaches indicates a much lower figure--in the range 40,000-100,000 genes. Renaturation kinetic analysis reveals the presence of many repetitive elements in the human genome. Some of these are quite simple and highly reiterated, others are more complex and exist in fewer copies. The distribution and function of some of these families and the evolution of complex genomes from simpler ones are discussed.

Chromosome Mapping↗

PubMed1990

Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.

Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.

Amino Acid Sequence↗

PubMed2004

Cross-complementation between the products of the genes P1 and ORF6 of Mycoplasma pneumoniae subtypes 1 and 2.

The genes P1 (MPN141) and ORF6 (MPN142) are essential for the successful colonization of the human respiratory tract by Mycoplasma pneumoniae. Both genes are located in the P1 operon, which consists of three genes. The P1 gene is the second gene in the operon, followed by the ORF6 gene. The P1 gene contains two (RepMP2/3, RepMP4) and the ORF6 gene one (RepMP5) specific repetitive DNA sequence, of which seven to nine similar but not identical copies are dispersed on the genome. Despite this large potential pool for genetic variation, M. pneumoniae isolates from patients contain only one of two distinct combinations of the genes P1 and ORF6. To analyse the functions of the repetitive DNA sequences, two 'new' combinations of the genes P1 and ORF6 were constructed, keeping the P1 gene constant but exchanging RepMP5 copies of the ORF6 gene. M. pneumoniae was transformed with these constructs and the transformants were tested for their ability to grow and survive under in vitro conditions and in guinea pigs. The two transformants colonized the respiratory tract of guinea pigs and showed no obvious differences in their growth behaviour compared to M. pneumoniae isolates from patients. The results indicate that the subtype-specific combinations of the repetitive elements in the P1 and ORF6 genes are not essential for the successful adherence of M. pneumoniae to host cells and the colonization of the respiratory tract of guinea pigs.

Adhesins, Bacterial↗

PubMed1992

Repetitive sequences cloned from Leptospira interrogans serovar hardjo genotype hardjoprajitno and their application to serovar identification.

We selected, from a genomic library of Leptospira interrogans serovar hardjo genotype hardjoprajitno, two probes containing repetitive sequences (pL1 and pL590). The hybridization patterns of these probes to DNA isolated from a variety of Leptospira serovars were examined and their ability to detect subtle differences at the genomic organization level was established. We identified the DNA fragments within pL1 and pL590 which are sufficient to yield polymorphic hybridization patterns; these results define the upper size limit of two novel repetitive elements in the Leptospira genome. The pattern and degree of hybridization observed for the serovars tested in this work were used to divide Leptospira spp. into groups which share genetic relatedness; our conclusions are consistent with previous classifications by other authors.

Blotting, Southern↗

PubMed2005

Novel variants of human SCaMC-3, an isoform of the ATP-Mg/P(i) mitochondrial carrier, generated by alternative splicing from 3'-flanking transposable elements.

CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/P(i) co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/P(i) carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3'-donor site and distinct 5'-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b', -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101-2111].

3' Flanking Region↗

PubMed1991

The Entamoeba histolytica rDNA episome: nuclear localization, DNAase I sensitivity map, and specific DNA-protein interactions.

Structural and functional features of the extrachromosomal DNA element that contains the ribosomal RNA genes of Entamoeba histolytica were studied using a variety of techniques. Using in situ hybridization, the element was found to be distributed along the inner phase of the nuclear membrane in the trophozoite stage; it appears to be part of the so-called peripheral chromatin. DNAase I-sensitive regions on the episome were mapped and found to correspond to the borders of the ribosomal RNA coding region. Other DNAase I-sensitive regions were found to correspond to DNA containing a 145bp sequence that exists in the episome as tandem repeats. Electrophoretic shift assays and footprinting experiments demonstrate the existence of specific nuclear factors that bind specifically to the 145bp repeat. Preliminary analysis of the binding factors showed that a 28 kDa polypeptide is a likely candidate for a specific DNA:protein interaction involving the repetitive element. These results suggest that a protein-binding domain within the 145bp repeat may have a specific function in the episome.

Animals↗

PubMed1996

Specific quantitation of genomic DNA in the femtogram range by amplification of repetitive sequences.

An assay was developed to specifically quantitate concentrations as low as 50 fg/ml genomic DNA based on the amplification of repetitive sequences. Reliable results were obtained by using internal standard molecules which were coextracted, coamplified, and coanalyzed with the nucleic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers followed by quantitation of fluorescence derived from the reaction products after separation by PAGE. Based on the known amount of added internal standard molecules and the intensities of the fluorescence of the reaction products, the primary results of the assay were obtained as copies per milliliter of sample. These were converted into mass units of DNA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA, and the sequences amplified were CR1 repetitive elements. This type of assay may be applied in cases where a sensitive and precise quantitation of genomic DNA is required, such as in the quality control of biological products.

Animals↗