Showing papers on "genomic DNA published in 1982"

Highly repeated sequences in mammalian genomes.

Abstract: Publisher Summary This chapter discusses the structure and organization of mammalian, highly repeated sequences at the molecular level. There is a description of tandemly repeated sequences, that are, satellites, and the segments that are interspersed among other genomic DNA sequences. The methods for the analysis of repeated DNA sequences are measurement of DNA renaturation kinetics and isopycnic centrifugation in gradients of CsCl and CsSO4. Eukaryote genomes can be divided into classes of DNA sequences according to the reiteration frequency. Many highly repeated sequences are in long tandem arrays. Some repetitive sequences are dispersed throughout major portions of genomes amid either other repeated sequences or sequences present only once per genome, that are, uniquesequences. The characteristic organizational feature of satellites and cryptic satellites is the tandem repetition of a unit DNA sequence. Satellite arrays resist separation by isopycnic centrifugation and instead remain within the main density fraction of genomic DNA. The repeat unit and its tandem organization can be revealed by restriction endonuclease digestion.

The Human Growth Hormone Gene Family: Nucleotide Sequences Show Recent Divergence and Predict a New Polypeptide Hormone

Abstract: The nucleotide sequences of three nonallelic human genomic DNA fragments which each contain one member of the growth hormone gene family are presented. These genes code for the known polypeptide hormones, growth hormone (hGH), chorionic somatomammotropin (hCS), and a yet unknown protein which differs from hGH in 13 positions. Each gene is structured into five exons, the four introns occurring at identical positions. Reflecting recent gene divergence, 90-95% sequence homology is seen in exons, introns, 5′, and immediate 3′ nontranscribed regions. The regions downstream of the polyadenylation sites in the genes for hGH and its variant, but not for hCS, contain members of an Alu family repeat sequence.

Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses.

Abstract: Harvey and Kirsten murine sarcoma viruses each encode a structurally and functionally related 21-kilodalton protein (p21), which is the transforming protein of each virus. Using probes from the 0.9-kilobase (kb) p21-coding region of each virus (called v-Ha-ras and v-Ki-ras, respectively), we have molecularly cloned from normal human genomic DNA the sequences that hybridize to these probes. Four evolutionarily divergent restriction endonuclease fragments were isolated. Two hybridized preferentially to v-Ha-ras and were designated human c-Ha-ras1 and c-Ha-ras2; two hybridized preferentially to v-Ki-ras and were called c-Ki-ras1 and c-Ki-ras2. Human c-Ha-ras1 contained 0.9 kb of sequence homologous with v-Ha-ras interspersed with three intervening sequences; this gene was closely related to a previously cloned rat c-Ha-ras gene that also contained intervening sequences. Human c-Ha-ras2 was more divergent from v-Ha-ras and also hybridized poorly to human c-Ha-ras1. One c-Ki-ras gene contained 0.9 kb homologous to v-Ki-ras and had one intervening sequence, whereas the other contained only 0.3 kb homologous to v-Ki-ras. The results indicated that human DNA contains several copies of the c-ras family and that c-Ha-ras1 (with intervening sequences) was more highly conserved evolutionarily than was c-Ha-ras2.

Cloning of antibiotic resistance and nutritional genes in streptomycetes.

Abstract: Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66 were adjusted to generate reproducibly a population of cloned genes likely to represent the entire genome. Factors which influence the recovery of viable transformants include: growth phase of the mycelium, ionic and osmotic characteristics of the medium during protoplast formation and transformation, and moisture content and protoplast density during regeneration. A modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes, using one of two plasmid vectors. Viomycin resistance genes on BamHI or PstI fragments were cloned from S. vinaceus genomic DNA into S. lividans, using the SLP1.2 vector. At least three different S. vinaceus BamHI fragments (1.9, 5.8, or 8.5 kilobases) confer viomycin resistance; only one PstI fragment (4.3 kilobases) was found. Recombinant plasmids were all able to produce lethal zygosis and to be transferred by conjugation within S. lividans. SCP2 was used to clone S. coelicolor A3(2) genes that "complemented" the auxotrophic mutation hisD3, argA1, or guaA1. Recombinant DNA technology can now be applied to economically and academically interesting problems unique to streptomycete molecular biology.

An analysis of cosmid clones of nuclear DNA from Trypanosoma brucei shows that the genes for variant surface glycoproteins are clustered in the genome

Abstract: Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment.

Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene.

Abstract: We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5 It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene

Characterization of the structural gene and putative 5′-regulatory sequences for human proopiomelanocortin

Abstract: It is now well established that the hormones corticotropin (ACTH) and β-lipotropin (β-LPH) are contained within a common precursor peptide called proopiomelanocortin (POMC), which is synthesized in the anterior and/or intermediate lobes of the pituitary1–3 and in the hypothalamus4. The recent cloning of a ‘full-size’ cDNA copy of bovine POMC mRNA has revealed the presence of additional peptides in the previously ‘cryptic’ segment of the precursor protein5. This cDNA has been used as a hybridization probe for the isolation and characterization of a bovine genomic DNA segment encoding the complete POMC mRNA6, and for isolation of part of the corresponding human genomic DNA sequence7. DNA sequence analysis of these isolates and of part of the rat POMC gene8 has shown that, in the species studied, certain segments of the POMC structural gene have been conserved during evolution, whereas the nucleotide sequences of other segments have diverged sharply. We have now isolated and characterized a 11.6-kilobase (kb) human genomic DNA fragment that encodes the complete POMC mRNA and putative regulatory sequences proximate to the gene (which is repressed by glucocorticoids in vivo9). We have also analysed the 800-base pair (bp) segment preceding the mRNA coding sequence, within which—at a position nearly 480 bp upstream from the mRNA start site—is a 21-bp segment that shares homology with a DNA sequence beginning 370 bp upstream from two other glucocorticoid-controlled genes. We speculate that these sequences may be involved in the regulation of POMC gene expression by glucocorticoids.

Base sequence of a cloned snake W-chromosome DNA fragment and identification of a male-specific putative mRNA in the mouse

Abstract: A 2.5-kilobase fragment of a sex-specific satellite DNA from the Colubrid snake species Elaphe radiata has been cloned, and its sequence has been determined. It contains 26 and 12 copies, respectively, of two base quadruplets, G-A-T-A and G-A-C-A, as its sole highly repetitious elements. Southern hybridization experiments with genomic DNA of the chicken, the mouse, and man indicated male sex-specific conservation of at least parts of this cloned DNA. In situ hybridization experiments with metaphase chromosomes of the mouse showed that elements that can cross-hybridize with parts of the cloned snake DNA are concentrated in the pericentric region of the Y chromosome. In blot hybridization experiments with liver poly(A)+ polysomal RNAs of male and female mice, a probe consisting of the first 1,224 bases of the cloned snake DNA singled out a male-specific RNA of 1,250-1,400 bases. Inasmuch as the proximal end of this probe contained an open reading frame (44 consecutive amino acid-specifying codons), the male-specific putative mRNA so detected may specify H-Y antigen. By contrast, a probe consisting of bases 1,480-1,906, containing the simple repeats of the quadruplets, singled out a shorter (approximately 1,000-base) RNA from males and females alike. Although this RNA is poly(A)+, we have yet to establish its attachment to ribosomes.

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

Abstract: pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.

Cloning and analysis of cDNAs encoding plant storage protein precursors

Abstract: The major storage protein in many legume seeds is legumin, a hexameric molecule of molecular weight (Mr) 360,000–400,000 consisting of six subunit pairs, each of which comprises a 40,000-Mr, acidic polypeptide linked by disulphide bonds to a 20,000-Mr, basic polypeptide1. We have recently shown that these subunit pairs are synthesized, in vivo and in vitro in Pisumand Vicia2,3, as a 60,000-Mr precursor polypeptide, putatively containing one copy each of the acidic and basic summits covalently linked together. We describe here the cloning and identification of cDNA sequences coding for the legumin precursor. Additionally, we have used these cDNAs to verify that the legumin mRNAs are of sufficient size to code for the precursor and to demonstrate the presence of four single-copy legumin genes in genomic DNA. Possible mechanisms for the processing of the legumin precursor are discussed. Identification of cloned cDNA sequences coding for vicilin, the secondary storage protein of Pisum sativum4, is also reported.

A + T-rich linkers define functional domains in eukaryotic DNA

Abstract: Although an increasing number of detailed DNA sequence studies are being carried out, little is known about the long-range organization of the eukaryotic genome in relation to chromosomal organization and to functional units of transcription, replication, meiotic crossing-over and nuclear architecture. Based on electron microscope (EM) observations of partially denatured DNA of duck, mouse and rat, we have recently reported a systematic punctuation of DNA by early melting zones (‘A + T-rich linkers’)1 ∼800 base pairs (bp) long which are either clustered or at intervals of 10–40 kbp. In an attempt to correlate this feature with the organization of specific genes, we examined cloned genomic DNA from chick and Drosophila, including the globin gene family, and an isolated high-molecular-weight mRNA. We show here that the chick α-globin gene cluster is framed by a cluster of A + T-rich linkers at the 5′ end and by an isolated linker at the 3′ end which maps close to the sites of transcription termination and DNase hypersensitivity. The resulting frame of ∼15 kbp correlates well with the maximum size of globin gene transcripts found at the pre-mRNA level. The β-globin gene cluster is also framed, 5′ by a single A + T-rich linker and 3′ a cluster of four linkers. In Drosophila, the single mRNA sequence of ∼3.3 kbp for the ecdysone-inducible P1 protein is similarly delimited by A + T-rich linkers. A + T-rich linkers thus seem to define domains of the eukaryotic genome which may correlate with putative functional units. The specific organization observed in total DNA of duck, mouse, rat, man, chicken and Drosophila is confirmed in gene-specific cloned genomic DNA.

A Gene Deletion is Responsible for Absence of Human Chorionic Somatomammotropin

Abstract: We have examined the human growth hormone (hGH) and human chorionic somatomammotropin (hCS) family of genes in genomic DNA from an individual with complete antenatal deficiency of hCS. Following digestion with a variety of bacterial restriction endonucleases, the DNA from this individual produced fewer fragments with homology to a radiolabeled hCS cDNA probe than did control DNA specimens. The patterns indicated that his DNA contained the normal hGH gene and an "hGH-like" gene, but lacked the hCS gene, a variant hGH gene, and another gene or genes with structural homology to hGH and hCS, which were present in all control DNA specimens. The findings were consistent with homozygosity for a gene deletion with a minimum length of 18.5 kb. Analysis of polymorphic restriction site variation related to the hGH and hCS gene cluster indicated that both parents and three older siblings were heterozygous for the deletion. The association between gene deletion and a normal growth pattern in this individual indicates ...

Two main groups of mouse major urinary protein genes, both largely located on chromosome 4.

Abstract: Fourteen different major urinary protein (MUP) genomic clones from BALB/c mice were isolated By restriction site mapping, six of these form two sets of three overlapping clones By the criterion of cross-hybridization, the 10 different genes fall into two groups of four (Group 1) and three (Group 2) genes, while three genes fall into neither group Southern blot analysis of genomic DNA with Group 1 and Group 2 plasmid subclones shows that the haploid mouse (BALB/c) genome contains approximately 15 Group 1 genes, 12 Group 2 genes and at least seven MUP genes that belong to neither group An analysis of mouse-Chinese hamster hybrid cell lines shows that most, if not all, Group 1 and Group 2 genes are located on mouse chromosome 4

General method for cloning amplified DNA by differential screening with genomic probes

Abstract: Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.

Isolation of cDNA clones encoding HLA-DR alpha chains.

Abstract: HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.

A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations

Abstract: Several genomic clones from Petroselinum hortense, Zea mays and Antirrhinum majus all homologous to cloned Petroselinum chalcone synthase cDNA were isolated using the λgt WES cloning system. Clones containing the chalcone synthase structural gene were identified by hybridization to cDNA from Petroselinum hortense, genomic wildtype, mutant and revertant DNA. Among the 5 different clones from Petroselinum hortense, PH3 is the most likely candidate to contain at least a portion of the chalcone synthase gene. None of the 4 Zea mays clones appeared to contain part of the chalcone synthase gene. Among the 2 different clones from Antirrhinum majus, AM3 contains the portion of the chalcone synthase structural gene which is altered in the mutant nivea recurrens (niv rec). This mutant is considered to be due to the integration of a transposable element. In revertants of niv rec to niv + the wildtype locus is restored molecularly.

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

Abstract: Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

Genomic clones of a wild-type allele and a transposable element-induced mutant allele of the sucrose synthase gene of Zea mays L.

Abstract: In an attempt to isolate the transposable genetic element Ds from Zea mays L, we cloned DNA fragments hybridizing to a cDNA clone derived from the sucrose synthase gene in a lambda vector (lambda::Zm Sh) The fragments cloned from wild-type and from the Ds-induced mutant sh-m5933 (lambda::Zm sh-m5933) share a segment 6 kb long while a contiguous segment of 15 kb of lambda::Zm sh-m5933 (mutant-derived DNA) does not hybridize to the DNA segment cloned from the wild-type Restriction maps are given, and the junction point between the two DNA segments in the mutant clone was determined Hybridization of DNA fragments, present in the wild-type DNA of lambda::Zm Sh, but not in the mutant clone, lambda::Zm sh-m5933, to genomic DNA of sh-m5933 showed that no part of this DNA is deleted It cannot be said whether the DNA found in the mutant, but not in the wild-type clone, has been brought there by Ds insertion or by another Ds-dependent DNA rearrangement The mutant-derived DNA was hybridized to genomic DNA of various maize lines digested by several restriction endonucleases Approximately 40 bands were detected The mutant-derived DNA contains two pairs of inverted repeats several hundred nucleotide pairs long, one of which is located at the junction to wild-type-derived DNA

Cloning of Two Genetically Transmitted Moloney Leukemia Proviral Genomes: Correlation Between Biological Activity of the Cloned DNA and Viral Genome Activation in the Animal

Abstract: The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10−5 XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10−9 XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10−7 PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.

Comparison of the hinge-coding segments in human immunoglobulin gamma heavy chain genes and the linkage of the gamma 2 and gamma 4 subclass genes.

Abstract: The genes for the human immunoglobulin heavy chain constant region subclasses have been examined in clones isolated from a phage library containing human genomic DNA Nucleotide sequencing and restriction enzyme mapping show that the CH1, hinge, CH2, and CH3 domains are present as distinct genetic elements separated by short intervening sequences in these genes: the gamma 3 gene is unique amongst these genes in that it possesses four separate hinge-coding segments A further gamma gene has been identified which does not correspond to a known human gamma protein: this gene contains a hinge related to the first hinge of the gamma 3 gene and may represent an inactive or pseudo gamma gene Comparison of a number of overlapping clones containing gamma 2 and gamma 4 genes shows that they are separated in human DNA by approximately 19 kb and that the gamma 2 gene is located upstream of gamma 4 Both genes (order 5'C gamma 2-C gamma 4-3') are orientated in the same direction of transcription