Showing papers on "genomic DNA published in 1988"

Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification

Abstract: The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.

Sequence and characterization of 6 Lea proteins and their genes from cotton.

Abstract: Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins. The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.

Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets.

Abstract: Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly----Asp) and one at the tip of the second zinc finger (Arg----Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.

Abstract: Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.

Genomic amplification with transcript sequencing.

Abstract: A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.

Mouse lysozyme M gene: isolation, characterization, and expression studies.

Abstract: We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels.

A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi.

Abstract: Fungal genetic studies require a rapid method of isolating DNA from a large number of samples for restriction enzyme analysis. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol35/iss1/11 Complementation studies including mts(MN9), cpc-1 (CD86 and j5) and mts(MN1) were performed as already described for mts MNl) (Koch and Bartheless, 1987). The mutants were recessive to their respective wild type alleles, but complementation of the amino acid analogue sensitive phenotype was not observed in heterocaryons carrying mutant alleles simultaneously. These findings suggest that cpc-1, mts(MN9) as well as mts(MNl) belong to the same complementation group. Institut für Angewandte Genetik, Universität Hannover, 3000 Hannover FRG. Supported by the Deutsche Forschungsgemeinschaft. Lee, S.B., M.G. Milgroom # Fungal genetic studies require a rapid method of isolating DNA from a large number of and J.W. Taylor samples for restriction enzyme analysis. Previous methods we have used are limited by relaA rapid, high yield mini-prep method tively low yield of 50 ug DNA/0.1g lyophilized mycelium (Zolan, M.E. and P.J. Pukkila 1986. Mol. for isolation of total genomic DNA Cell. Biol. 6:195-200) or tedious gel exclusion column chromatography (Biel, S.W. and F.W. from fungi. Parrish 1986. Anal. Biochem. 154:21-25). In addition, these two methods yielded no readily digestable DNA from Phytophthora cinnamomi. The following method facilitates rapid isolation of large quantities of easily digested total, genomic DNA from several species of Phytophthora, including P. cinnamomi, and several species of Boletus, Chroogomphus vinicolor, Gomphidius glutinosus, Leccinum manzanitae, Magnaporthe grisea, Neurospora crassa, N. tetrasperma, Omphalotus olivascens, and Talaromyces flavus. isolation of DNA from two Yield was increased to 200 ug DNA/0.1 g lyophilized mycelium and to three times as many samples can be achieved using this rapid method (current record is 64 isolates in one day versus 24 using previous methods). DNA has been successfully cut with all restriction enzymes tried to date. Solutions needed: 1. Lysis buffer: 50 mM Tris-HCl 50 mM EDTA 3% SDS 1% 2-mercaptoethanol (add just before use) 2. Chloroform:phenol (1:1) 3. SEVAG (chloroform:isoamyl alcohol, 24:1) 4. 3 M NaOAc (pH 8.0) 5. Isopropanol 6. Ethanol (100%, -20°C)

Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene.

Abstract: The immediate-early IE-1 gene of Autographa californica nuclear polyhedrosis virus was cloned, and its nucleotide sequence was determined. Sequence analysis indicated that this gene would encode a protein of 582 amino acids with a predicted molecular weight of 66,822. Analysis of IE-1 gene expression during baculovirus infection identified two transcripts. One, 1.9 kilobases (kb), was expressed at constant steady-state levels throughout infection, whereas the other, 2.1 kb, was expressed only early in infection. Analysis of IE-1 cDNA clones demonstrated that the 2.1-kb transcript contained the entire 1.9-kb transcript (exon 1) plus an additional 5' end (exon 0). Genomic Southern analysis placed the exon 0 sequences on the EcoRI B fragment, 4 kilobase pairs upstream of exon 1. Sequencing of the upstream region identified an open reading frame whose 5' end was identical to the exon 0 sequences in the cDNAs. Examination of the genomic DNA sequences around the exon-exon junction revealed sequences similar to published consensus splice acceptor and donor sequences. This is the first example of splicing of any viral transcript during baculovirus infection.

Direct sequencing of enzymatically amplified human genomic DNA.

Abstract: The polymerase chain reaction is a recently described technique that uses flanking oligonucleotide primers and repeated cycles of enzymatic primer extension to amplify a short segment of DNA by greater than 100,000-fold. By use of sequencing primers located internal to the amplification primers, direct genomic sequence was obtained from enzymatically amplified DNA by using the dideoxynucleotide chain-termination method. The method is relatively simple and offers significant advantages in identifying mutations in genes for which the normal sequence is known. Heterozygous and homozygous mutations in the human beta- and gamma-globin loci were unambiguously identified in 3 days with less than 1 microgram of genomic DNA.

Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames.

Abstract: The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICP0 and is transcribed in the opposite direction from ICP0, producing LR-RNAs that are complementary (antisense) to ICP0 mRNA. The LR gene is thought to be involved in HSV-1 latency. We report here the fine mapping and partial sequence analysis of this HSV-1 LR gene. 32P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LR-RNA 2, respectively. Their 5' ends started approximately 1,210 nucleotides downstream from the 3' end of the ICP0 mRNA. Their 3' ends overlapped ICP0 by nearly 1,000 nucleotides. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.

Enhancer-like interferon responsive sequences of the human and murine (2'-5') oligoadenylate synthetase gene promoters.

Abstract: The human (2'-5') oligo(A) synthetase gene contains two independent cis-acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN-inducibility to the herpes tk promoter. Two murine (2'-5') oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK-CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon-responsive sequence (E-IRS) were cloned in 5' of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E-IRS. This binding is competed by the addition of cold synthetic mouse E-IRS or fragments of genomic DNA containing the E-IRS.

Characterization of the gene for the a subunit of human factor XIII (plasma transglutaminase), a blood coagulation factor

Abstract: Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a glycoprotein that circulates in blood as a tetramer (a2b2) consisting of two a and two b subunits. The primary structures of the a and b subunits of human factor XIII have been reported by a combination of cDNA cloning and amino acid sequence analysis. To establish the gene structure of the a subunit for factor XIII, several human genomic libraries were screened by using the cDNA encoding the a subunit as a probe. Among approximately equal to 5 x 10(7) recombinant phage, 121 have been shown to contain an insert encoding a portion of the a subunit. Twenty-five unique clones were then characterized by restriction mapping, Southern blotting, and DNA sequencing. Overlapping clones encoding the a subunit of factor XIII span greater than 160 kilobases. The gene was found to contain 15 exons separated by 14 introns. All the sequences of the introns at the intron-exon boundaries were GT-AG, which are the same as those found in other eukaryotic genes. DNA sequence analysis revealed that the activation peptide released by thrombin, the active site cysteine region, the two putative calcium-binding regions, and the thrombin cleavage site leading to inactivation are encoded by separate exons. This suggests that the introns may separate the a subunit into functional and structural domains. A comparison of the amino acid sequence deduced from the genomic DNA sequence with those deduced from cDNA or determined by amino acid sequence analysis of the plasma and placental proteins revealed apparent amino acid polymorphisms in six positions of the polypeptide chain of the a subunit.

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types.

Abstract: Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.

Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.

Abstract: The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

Human retinoblastoma susceptibility gene: genomic organization and analysis of heterozygous intragenic deletion mutants.

Abstract: A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a restriction and exon map of the RB gene was constructed by aligning overlapping genomic clones, yielding three contiguous regions ("contigs") of 150 kilobases total length separated by two gaps. At least 20 exons were identified in genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origins of their shortened RB mRNA transcripts. The same probe detected genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastoma. Third, this probe also detected a polymorphic site for BamHI with allele frequencies near 0.5/0.5. Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families.

LR1: a candidate RNA virus of Leishmania.

Abstract: Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately 6000 nucleotides, is single-stranded, and is largely, if not exclusively, located in the cytoplasm. No homologous LR1 sequences are detected in genomic DNA. The candidate viral RNA is associated with a spherical particle 32 nm in diameter that has a sedimentation coefficient of approximately 130 S. There is as yet no evident effect of this potential virus on parasite physiology or the disease caused by the parasite.

Structure of the human ornithine transcarbamylase gene

Abstract: Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.