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At least 1,135 records · Page 63Linked to original sources
PubMed1993

Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus by using universal primers.

Fast acquisition of unknown nucleotide sequences around a known sequence has important implication in molecular biology, especially in genome mapping. We have developed a method, termed restriction site polymerase chain reaction (RS-PCR), that utilizes specially designed primers that recognize, anneal, and sustain PCR. These primers, termed restriction site oligonucleotides (oligonucleotide primers specific for a given restriction enzyme recognition sequence or RSOs), could be generated corresponding to any restriction enzyme irrespective of the length of the recognition site and used as PCR primers corresponding to the unknown region of a DNA segment. In this method a first round of PCR is carried out in different tubes with a set of RSOs and a primer specific to the known region. A second round of PCR is then performed on the products of the first PCR with the same RSOs and another specific primer internal to the first one. Subsequently, the products of the last round of PCR are transcribed with an appropriate RNA polymerase and sequenced with a reverse transcriptase with an end-labeled specific primer internal to the second specific PCR primer. To demonstrate the applicability of RS-PCR in retrieving unknown sequences around a known sequence, we have used a set of four RSOs and three specific primers representing the known sequence and have successfully obtained hitherto unknown factor IX sequences (12 of 12 times) from three species starting from genomic DNA. The sequences obtained indicate the presence of a conserved stretch of 20 nucleotides in the 3' noncoding region of the factor IX gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Organization and evolution of a gene-rich region of the mouse genome: a 12.7-Mb region deleted in the Del(13)Svea36H mouse.

Del(13)Svea36H (Del36H) is a deletion of approximately 20% of mouse chromosome 13 showing conserved synteny with human chromosome 6p22.1-6p22.3/6p25. The human region is lost in some deletion syndromes and is the site of several disease loci. Heterozygous Del36H mice show numerous phenotypes and may model aspects of human genetic disease. We describe 12.7 Mb of finished, annotated sequence from Del36H. Del36H has a higher gene density than the draft mouse genome, reflecting high local densities of three gene families (vomeronasal receptors, serpins, and prolactins) which are greatly expanded relative to human. Transposable elements are concentrated near these gene families. We therefore suggest that their neighborhoods are gene factories, regions of frequent recombination in which gene duplication is more frequent. The gene families show different proportions of pseudogenes, likely reflecting different strengths of purifying selection and/or gene conversion. They are also associated with relatively low simple sequence concentrations, which vary across the region with a periodicity of approximately 5 Mb. Del36H contains numerous evolutionarily conserved regions (ECRs). Many lie in noncoding regions, are detectable in species as distant as Ciona intestinalis, and therefore are candidate regulatory sequences. This analysis will facilitate functional genomic analysis of Del36H and provides insights into mouse genome evolution.

Animals↗
PubMed2003

Epigenetic dynamics of imprinted X inactivation during early mouse development.

The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo.

Acetylation↗
PubMed1989

Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.

Animals↗
PubMed1997

Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with a characteristic pattern of visceromegaly and predisposition to childhood tumours. BWS is a genetically heterogeneous disorder; most cases are sporadic but approximately 15% are familial and a small number of BWS patients have cytogenetic abnormalities involving chromosome 11p15. Genomic imprinting effects have been implicated in familial and non-familial BWS. We have investigated the molecular pathology of 106 sporadic BWS cases; 17% (14/83) of informative cases had uniparental disomy (UPD) for chromosome 11p15.5. In each case UPD appeared to result from a postzygotic event resulting in mosaicism for segmental paternal isodisomy. The critical region for isodisomy was refined to a 25 cM interval between D11S861 and D11S2071 which contained the IGF2, H19, and p57(KIP2) genes. In three cases isodisomy for 11q markers was detected but this did not extend further than 11q13-q21 suggesting that complete chromosome 11 disomy may not produce a BWS phenotype. The allele specific methylation status of the H19 gene was investigated in 80 sporadic BWS cases. All 13 cases with UPD tested displayed hypermethylation consistent with an excess of paternal H19 alleles. In addition, five of 63 (8%) cases with normal biparental inheritance had H19 hypermethylation consistent with an "imprinting centre" mutation (ICM) or "imprinting error" (IE) lesion. The phenotype of patients with putative ICM/IE mutations was variable and overlapped with that of non-UPD sporadic BWS cases with normal H19 methylation. However, exomphalos was significantly (p < 0.05) more common in the latter group. These findings may indicate differential effects on the expression of imprinted genes in chromosome 11p15 according to the precise molecular pathology. Analysis of H19 methylation is useful for the diagnosis of both UPD or altered imprinting in BWS and shows that a variety of molecular mechanisms may cause relaxation of IGF2 imprinting in BWS.

Aneuploidy↗
PubMed1990

Organization of the 5S rRNA genes in the soybean Glycine max (L.) Merrill and conservation of the 5S rDNA repeat structure in higher plants.

The 5S rRNA gene of the soybean Glycine max (L.) Merr. has been cloned on a 556-bp fragment of DNA and sequenced. This fragment contains two copies of the soybean 5S rDNA sequence, one intact and one truncated, separated by noncoding DNA. We have used this clone to investigate the organization of the 5S genes within the soybean genome and the extent of their methylation. Our results demonstrate that soybean 5S genes are clustered, organized into tandem repeats of 330 bp, and extensively methylated. Hybridization of the 5S sequence to Southern transfers of soybean DNA digested with BamHI reveals a striking ladderlike pattern. Hybridization of the soybean 5S sequence to a wide variety of plant DNAs results in similar patterns, suggesting that the 5S rDNA sequence, gene organization, and methylation pattern are conserved in many higher plants.

Base Sequence↗
PubMedDate not supplied

Isolation and characterization of the Drosophila ornithine decarboxylase locus: evidence for the presence of two transcribed ODC genes in the Drosophila genome.

The polymerase chain reaction (PCR) was used to isolate two Drosophila ornithine decarboxylase (ODC) genes. Two mixtures of degenerate oligonucleotides corresponding to peptides that are fully conserved among ODCs from widely diverged species were used as opposing primers in the PCR with cDNA or genomic DNA as templates. Sequence analysis of the resulting DNA products confirmed their identity as ODC fragments. The genomic PCR product was then used as a probe for screening a Drosophila genomic library, resulting in the isolation of genomic clones representing two distinct ODC genes (dODC1 and dODC2). Sequence analysis of both genes demonstrated that although varying at their coding and noncoding regions, their overall structure is extremely similar containing 6 exons and 5 short introns. Southern blot and sequence analyses revealed that the two ODC genes are arranged in a tandem head-to-tail configuration. Both ODC genes were assigned by in situ hybridization analysis to position 44A on the right arm of the second chromosome. The isolation of cDNA clones corresponding to these two ODC genes demonstrated that both are transcribed in the adult fly. We hope that the isolation of genomic and cDNA clones of Drosophila ODC will permit the investigation of the expression of ODC during Drosophila development and the role of polyamines in this process.

Amino Acid Sequence↗
PubMed2006

Transcriptional and posttranscriptional regulation of transcription factor expression in Arabidopsis roots.

Understanding how the expression of transcription factor (TF) genes is modulated is essential for reconstructing gene regulatory networks. There is increasing evidence that sequences other than upstream noncoding can contribute to modulating gene expression, but how frequently they do so remains unclear. Here, we investigated the regulation of TFs expressed in a tissue-enriched manner in Arabidopsis roots. For 61 TFs, we created GFP reporter constructs driven by each TF's upstream noncoding sequence (including the 5'UTR) fused to the GFP reporter gene alone or together with the TF's coding sequence. We compared the visually detectable GFP patterns with endogenous mRNA expression patterns, as defined by a genome-wide microarray root expression map. An automated image analysis method for quantifying GFP signals in different tissues was developed and used to validate our visual comparison method. From these combined analyses, we found that (i) the upstream noncoding sequence was sufficient to recapitulate the mRNA expression pattern for 80% (35/44) of the TFs, and (ii) 25% of the TFs undergo posttranscriptional regulation via microRNA-mediated mRNA degradation (2/24) or via intercellular protein movement (6/24). The results suggest that, for Arabidopsis TFs, upstream noncoding sequences are major contributors to mRNA expression pattern establishment, but modulation of transcription factor protein expression pattern after transcription is relatively frequent. This study provides a systematic overview of regulation of TF expression at a cellular level.

Arabidopsis↗
PubMed2001

CTCF, a candidate trans-acting factor for X-inactivation choice.

In mammals, X-inactivation silences one of two female X chromosomes. Silencing depends on the noncoding gene, Xist (inactive X-specific transcript), and is blocked by the antisense gene, Tsix. Deleting the choice/imprinting center in Tsix affects X-chromosome selection. Here, we identify the insulator and transcription factor, CTCF, as a candidate trans-acting factor for X-chromosome selection. The choice/imprinting center contains tandem CTCF binding sites that function in an enhancer-blocking assay. In vitro binding is reduced by CpG methylation and abolished by including non-CpG methylation. We postulate that Tsix and CTCF together establish a regulatable epigenetic switch for X-inactivation.

Animals↗
PubMed1990

Genomic sequences of human protamines whose genes, PRM1 and PRM2, are clustered.

Protamines are small, arginine-rich proteins involved in the condensation of sperm chromatin. Using cDNA clones, we have isolated the genes for both human protamines, i.e., protamine 1 (PRM1) and protamine 2 (PRM2), from a human cosmid library. Each of these genes contains a single intron consisting of 91 and 163 bp, respectively. From the 5'-noncoding region of PRM1 664 bp and from the 5'-noncoding region of PRM2 902 bp were determined. Both genes contain typical TATAA and CAAT boxes at conventional distances from the transcription start points, which by using primer extension experiments could be assigned to nucleotides -91 and -110 for PRM1 and PRM2 genes, respectively. Comparison of the 5'-noncoding regions of PRM1 and PRM2 genes reveals 12 different motifs in common, 8 of which are clustered in both genes and could reflect regulatory elements for testis- and spermatid-specific gene expression. Both human genes have been found to be clustered at a distance of 4.8 kb. Comparison of the genomic organization of human and mouse protamine genes revealed greater similarities between the two in the 5'-noncoding region.

Amino Acid Sequence↗
PubMed2002

Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain.

Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene delivery into the central nervous system (CNS). However, host inflammatory and immune responses may play a critical role in limiting the use of rAAV vectors for gene therapy and functional genomic studies in vivo. Here, we evaluated the effect of repeated injections of five rAAV vectors expressing different genetic sequences (coding or noncoding) in a range of combinations into the rat brain. Specifically, we wished to determine whether a specific immune or inflammatory response appeared in response to the vector and/or the transgene protein after repeated injections under conditions of mannitol coinjection. We show that readministration of the same rAAV to the CNS is possible if the interval between the first and second injection is more than 4 weeks. Furthermore, our data demonstrate that rAAV vectors carrying different genetic sequences can be administered at intervals of 2 weeks. Our data therefore suggest that the AAV capsid structure is altered by the vector genetic sequence, such that secondary structures of the single-stranded genome have an impact on the antigenicity of the virus. This study provides guidelines for more rational design of gene transfer studies in the rodent brain and, in addition, suggests the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

Animals↗
PubMed1985

A new member of the polyomavirus family: the hamster papovavirus. Complete nucleotide sequence and transformation properties.

The hamster papovavirus (HapV) is associated with multiple skin epitheliomas of the Syrian hamster. We have sequenced its genome. It is a double-stranded circular DNA of 5366 bp. The hypothetical genomic organization deduced from this nucleotide sequence is clearly of the polyoma type with the two strands coding in the opposite directions from a noncoding region that shows some of the features of a replication origin and a transcription control region. The amino acid sequences predicted from the open reading frames show an average of 50% homology with polyoma-coded polypeptides. The HapV is, after polyoma, the second example of a papovavirus coding for a middle T antigen. The cloned DNA can immortalize primary rat embryo cells and transform an established rat cell line. The viral DNA is stably integrated into the host genome.

Animals↗
PubMed2006

Repetitive DNA elements as mediators of genomic change in response to environmental cues.

There is no logical or theoretical barrier to the proposition that organismal and cell signaling could transduce environmental signals into specific, beneficial changes in primary structure of noncoding DNA via repetitive element movement or mutation. Repetitive DNA elements, including transposons and microsatellites, are known to influence the structure and expression of protein-coding genes, and to be responsive to environmental signals in some cases. These effects may create fodder for adaptive evolution, at rates exceeding those observed for point mutations. In many cases, the changes are no doubt random, and fitness is increased through simple natural selection. However, some transposons insert at specific sites, and certain regions of the genome exhibit selectively and beneficially high mutation rates in a range of organisms. In multicellular organisms, this could benefit individuals in situations with significant potential for clonal expansion: early life stages or regenerative tissues in animals, and most plant tissues. Transmission of the change to the next generation could occur in plants and, under some circumstances, in animals.

Animals↗
PubMed1991

Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment.

We have isolated and sequenced the gene encoding human tyrosinase, the key enzyme in pigment biosynthesis. The human tyrosinase gene contains five exons and spans more than 50 kb of DNA on chromosome segment 11q14----q21. We have also isolated a second segment in the human genome that is closely related to tyrosinase. The tyrosinase-related segment, located on 11p11.2----cen, contains only exons 4 and 5 plus adjacent noncoding regions. This segment is present in all human ethnic groups analyzed, and the noncoding nucleotide sequences shared by the 11q tyrosinase gene and the 11p tyrosinase-related segment differ by only 2.6%. This suggests that this segment of the tyrosinase gene was duplicated approximately 24 million years ago.

Amino Acid Sequence↗
PubMed1996

Multiple and tissue-specific promoter control of gonadal and non-gonadal prolactin receptor gene expression.

Prolactin receptors (PRLRs) are widely expressed, and multiple mRNA transcripts encoding PRLRs are present in prolactin target tissues. The molecular basis for the control of the PRLR gene expression is currently unknown. Analyses of the 5'-untranslated regions of PRLR mRNAs expressed in gonadal and non-gonadal tissues and their genomic organization revealed three alternative first exons designated as E11, E12, and E13. Each of these exons is alternatively spliced to a common noncoding exon (exon 2, nucleotides -115 to -56) that precedes the third exon containing the translation initiation codon. Alternative utilization of exons E11, E12, and E13, as well as alternative splicing of exon 2, generates multiple 5'-untranslated regions in PRLR transcripts. These alternative first exons (E11, E12, and E13) were found to be utilized in a tissue-specific manner in vivo. E11 is predominantly expressed in the ovary, E12 is specifically expressed in the liver, and E13 is expressed as a predominant form in the Leydig cell and as a minor form in the ovary and liver. Genomic 5'-flanking regions containing the three putative PRLR gene promoters (PI, PII, and PIII) that initiate the transcription of E11, E12, and E13, respectively, were identified. E11 was found to initiate from a single site at -549, E12 from multiple sites at -405, -461, and -506, and E13 from two major sites at -340 and -351. These findings indicate that multiple promoters control transcription of the PRLR gene and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues.

Alternative Splicing↗
PubMed2004

Intraspecies sequence comparisons for annotating genomes.

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intraspecies sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents, and a set of genomic intervals were amplified, resequenced, and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C. intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom. It also raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species.

Animals↗