Random DNA Fragments Research Articles

Assembly of the mitochondrial membrane system. Nucleotide sequence of a yeast nuclear gene (CBP1) involved in 5' end processing of cytochrome b pre-mRNA.

A nuclear gene (CBP1) of Saccharomyces cerevisiae involved in 5' end processing of the cytochrome b pre-mRNA has been cloned and sequenced. The gene was originally selected by transformation of cbp1 mutants with a recombinant plasmid bank of random fragments of yeast nuclear DNA ligated to a plasmid vector with autonomous replicative function in yeast. The recombinant plasmid pG60/T10 with a nuclear DNA insert of 6.7 kilobase pairs (kb) was used to construct a new plasmid pool with the CBP1 gene on smaller fragments of nuclear DNA. A number of subclones have been isolated with plasmids carrying inserts of 2.4 to 6.1 kb. The plasmid designated pG60/T31 confers respiratory competency to cbp1 mutants and restores their ability to synthesize mature cytochrome b mRNA. The pG60/T31 plasmid has a nuclear DNA insert of 2.4 kb. The sequence of the cloned fragment reveals only one open reading frame capable of coding for a protein. The reading frame is 1962 nucleotides long and codes for a basic polypeptide with a molecular weight of 76,140. A transcript of a size commensurate with the length of the gene has been detected in wild type yeast.

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii.

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 bp all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 bp that may play a role in promoting autonomous replication in C. reinhardii.

DNA insertions which affect the expression of the yeast iso-2-cytochrome c gene

The plasmid YCpCYC7(2) was constructed containing the Saccharomyces cerevisiae CYC7 gene, encoding the iso-2-cytochrome c protein, replicative sequences and selective markers from both E. coli and yeast, and the centromere of yeast chromosome III. The expression of the plasmid-CYC7 gene in yeast was similar to the low level expression characteristic of the chromosomal CYC7 gene. A number of insertions into the sequences 5' to the gene were constructed in vitro. The insertion at 142 by 5' to the coding sequence of a 400 by fragment which lies 5' to the CYC1 gene and is known to be essential for the high rates of CYC1 transcription increased transcription of the CYC7 gene to levels characteristic of CYC1 transcription. On the other hand, the insertion of random DNA fragments at the same position gave mostly decreased CYC7 transcription. In addition to these in vitro constructions, a mutant plasmid was selected which had increased CYC7 transcription. This mutation was caused by the insertion of the bacterial IS1 element 313 by 5' to the CYC7 coding sequence. The significance of these results is discussed in terms of two alternative models for CYC7 gene expression.

Tandem gene amplification mediates copper resistance in yeast.

Resistance to copper's toxicity in yeast is controlled by the CUP1r locus. This gene was cloned by transforming sensitive recipients (cup1(8)) with a collection of hybrid DNA molecules, consisting of random yeast DNA fragments inserted into the vector YRp7. Four resistant transformants were studied in detail. Autonomously replicating or integrated by homologous recombination into chromosomal sites, the corresponding plasmids and several subclones confer resistance on sensitive recipients carrying the natural variant allele, cup1(8). Tetrad analysis and genetic mapping established that integration occurs typically at the cup1(8) site located 28 centimorgans distal to thr1, a chromosome VIII marker. Restriction endonuclease cleavage and electrophoretic mobility studies revealed that the CUP1r locus consists of a tandem array of repetitive units. Each unit is 1.95 kilobases in length and contains single sites for Kpn I and Xba I and two Sau3A sites. The sensitive allele represents one repeat and the resistant allele embraces 15 tandemly arrayed repeat units. Progressive selections in higher copper concentrations establish strains with markedly enhanced resistance. Resistance, we propose, is mediated by a gene amplification mechanism based on unequal sister chromatid exchange.

Selective cloning of ColE1 DNA initiation sequences using the cloning vector M13ΔE101

DNA sequences required to direct single-strand to double-strand conversion by a øX174-type mechanism have been detected in ColEl plasmid DNA. The sites responsible for this DNA strand initiation, named rriA and rriB, have been localized to the L strand of the HaeII-E fragment and to the H strand of the HaeII-C fragment of ColE1. To define rriA and rriB more precisely, random fragments of ColEl DNA have been cloned into the single-stranded phage cloning vector M13ΔE101. This phage contains a viable deletion of the M13 complementary strand origin and makes small turbid plaques. Cloning of DNA initiation determinants into the unique EcoRI site of M13ΔE101 rescues the mutant phage and restores the ability to form clear plaques. DNA sequence analysis of the random, 100–400 bp inserts in clear plaque isolates has localized the rriA and rriB determinants within DNA fragments of about 100 bp. These initiation determinants promote assembly of a functional, multiprotein priming complex, “primosome”, identical to that utilized in the in vitro conversion of φX174 single-stranded DNA to the duplex replicative form.

Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration have been studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b have been assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene has been designated CBP1. A representative mutant from complementation group 60 (N5-26) has been transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation has been shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.

Cloning of the yeast methionyl-tRNA synthetase gene.

A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast Escherichia coli plasmid ((pFL1) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine. In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomic inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type. Measurement of the Km for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the Km for methionine to the same level as found for the wild type methionyl-tRNA synthetase. Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase. They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids

Separation of random fragments of DNA according to properties of their sequences.

The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.

Transformation in yeast: Development of a hybrid cloning vector and isolation of the can1 gene

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2μ circle. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.

Random location of nucleosomes on genes for 5 S rRNA.

The location of nucleosomes on genes for 5 S rRNA in rat liver was determined by the preparation of nucleosome core DNA fragments, hybridization with 5 S rRNA, RNase digestion, and gel electrophoresis. The resulting size distribution of RNA fragments was essentially the same as that found when the experiment was carried out with random DNA fragments.

Isolation of the chicken β-globin gene and a linked embryonic β-like globin gene from a chicken DNA recombinant library

A library of random chicken DNA fragments, 15–22 kb long, has been prepared in the vector λ Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, λ Chicken β-globin 1 (λCβG1), contains the adult chicken β-globin gene and a closely linked embryonic β-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5′ to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to λCβG1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian β-globin genes. The adult β-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian β-globin genes. Restriction enzyme analysis of the adult β-globin gene on λCβG1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the β-globin amino acid sequence as do the corresponding mammalian intervening sequences.

Overlap hybridization screening: Isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III

A set of four plasmids containing overlapping segments comprising a total of about 30 kbp of cloned DNA from chromosome III of yeast ( Saccharomyces cerevisiae) has been isolated and characterized by restriction endonuclease analyses and DNA: DNA hybridizations. Colony hybridization was carried out with labeled pYe( leu2)10, a plasmid carrying the yeast leu2 gene, to a bank of bacterial colonies containing recombinant plasmids constructed from the vector ColE1 and random fragments of yeast DNA. This resulted in the detection of two plasmids, pYe11G4 and pYe40C3, with DNA inserts which partially overlap the original cloned segment and contain additional DNA extending in opposite directions on the chromosome. By carrying out a second round of colony hybridization with pYe40C3, the cloned region was further extended in one direction. A region of DNA that is repeated at least ten times in the yeast genome was identified by hybridization of pYe11G4 to an EcoRI digest of total yeast DNA. The procedure described in this paper should allow the isolation of large sections of chromosomes, including non-transcribed regions, surrounding cloned genes.

The isolation and characterization of linked δ- and β-globin genes from a cloned library of human DNA

A cloned library of large, random embryonic human DNA fragments was constructed and screened for β-globin sequences using the cloned human β-globin cDNA plasmid pJW102 (Wilson et al., 1978) as a hybridization probe. Two independent clones were obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing. Each of the clones carries both the adult δ- and β-globin genes. The two genes are separated by approximately 5.4 kilobases (kb) of DNA and their orientation with respect to the direction of transcription is 5′-δ-β-3′. Both the δ-and β-globin genes contain a large noncoding intervening sequence (950 and 900 bp, respectively) located between the codons for amino acids 104 (arginine) and 105 (leucine). Although the location of the large intervening sequence within the coding regions of the two genes is identical, the two noncoding sequences bear little sequence homology. A second, smaller intervening sequence similar to that found in other mammalian β-globin genes was detected near the 5′ end of the human β-globin gene. The two independently isolated β-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the δ-globin gene of one of the clones. This suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the Pst I restriction enzyme recognition sequence.

A cloned Drosophila DNA fragment which codes for a 4 S RNA species

A collection of random Drosophila melanogaster DNA fragments cloned individually in Escherichia coli was screened for the presence of sequences complementary to the 4 S, 5 S and 5.8 S RNA species produced in the D. melanogaster K c tissue culture line. Four D. melanogaster DNA fragments were found which possessed sequences complementary to the 4 S RNA species but not complementary to the 5 S or 5.8 S RNA. One such cloned fragment (6.81 kilobase in length) was characterized further. It hybridizes in situ to region 22A-C of the left arm of chromosome 2 and does not contain repetitive sequences detectable by renaturation ( c 0 t) analysis. This same region was reported earlier by Steffensen and Wimber (Genetics (1971) 69, 163–178) to hybridize in situ to bulk tRNA extracted from D. melanogaster.

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of β-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked β-globin genes.

Experimental gene delivery systems for mammalian cells-the polyoma pseudovirus system

Polyoma pseudovirions consist of fragments of mouse DNA encapsidated within polyoma virus coats. The pseudovirions are adsorbed to and enter secondary mouse embryo or human embryo cells. The nuclear fraction of such pseudovirus-infected cells can be shown to contain pseudovirions as well as uncoated naked pseudoviral DNA. Thus, polyoma pseudovirions can deliver DNA to the nuclei of such mouse or human cells. Polyoma pseudovirions may be analogous to generalized transducing phage but as yet evidence for the expression of any genetic information of the pseudovirions is lacking. Since the DNA of polyoma pseudovirions appears to consist of random fragments of mouse DNA, a research program to learn how to assemble a gene-enriched particle has been started. A pancreatic DNase-resistant and high salt-resistant particle has been formed after the incubation of polyama empty capsids and DNA. This new particle has been isolated and characterized. Such experiments are continuing in order to develop polyoma pseudovirions as a gene delivery system for mammalian cells as well as whole animals.