Sequence variation in dispersed repetitive sequences in Saccharomyces cerevisiae

Two new dispersed repetitive DNA sequences related to the transposable element Tyl have been isolated from the genome of Saccharomyces cerevisiae. One sequence, designated Tyl-17, is present at about six copies per haploid genome, and one copy is located approximately 1000 base-pairs from the LEU2 locus on chromosome III. Tyl-17 is about the same size as Tyl (Cameron et al., 1979) and is flanked by δ sequences, but differs from Tyl by the presence of two large substitutions representing about 50% of the sequence. Tyl and Tyl-17 are found in a ‘head-to-head’ array in at least one cloned region of the yeast genome. Another sequence, designated Tyl-161, is situated about 9000 base-pairs from the PGK locus of chromosome III, and is structurally identical to Tyl except for the presence of a 1200 base-pair insertion near one end of the sequence element.     

Delta sequences and double symmetry in a yeast chromosomal replicator region

A specific autonomously replicating sequence (ars2) occurring near the ARG4 locus on chromosome VIII of yeast (Saccharomyces cerevisiae) has been localized on a 100 base-pair region of DNA. The nucleotide sequence of a 1517 bp² fragment spanning the ars2 region has been determined. The 100 bp ars2 region contains an 18 bp A + T sequence of perfect double symmetry (alphabetic symmetry superimposed on dyad symmetry). A 0.3 kb repetitive sequence (solo delta sequence) and a tRNA^Gln gene are located within the 1517 bp fragment along with ars2. The 100 bp ars2 region may be located within a divergent delta sequence that is oriented in an inverted and nearly tandem position with respect to the solo delta sequence.     

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

DNA sequences flanking an E. coli insertion element IS2 in a cloned yeast TRP5 gene

The insertion of an Escherichia coli IS2 element upstream from a cloned yeast TRP5 gene results in an increased level of active tryptophan synthase in trpAB E. coli host cells. This insertion occurs about 60 bp upstream from the first AUG of the TRP5 gene and is associated with a duplication of the sequence TTACA at the target site. The nucleotide sequence corresponding to the first 173 amino acids of the yeast TRP5 gene has also been determined. The N-terminal region of the yeast tryptophan synthase includes areas of strong homology with the alpha-subunit of the corresponding E. coli enzyme. Sequences from the 5' untranslated region upstream from the TRP5 gene are compared to homologous areas of other yeast genes.     

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts: Recombinant plasmid; sequence homology; stem and loop structure

The complete nucleotide sequence of a 16S ribosomal RNA gene from tobacco chloroplasts has been determined. This nucleotide sequence has 96% homology with that of maize chloroplast 16S rRNA gene and 74% homology with that of Escherichia coli16S gene.The 3′ terminal region of this gene contains the sequence ACCTCC which is complementary to sequences found at the 5′ termini of prokaryotic mRNAs.The large stem and loop structure can be constructed from the sequences surrounding the 5′ and 3′ ends of the 16S gene. These observations demonstrate the prokaryotic nature of chloroplast 16S rRNA.     

Sequence homology near the center of the extrachromosomal rDNA palindrome in Tetrahymena

The nucleotide sequence in the central region of the extrachromosomal ribosomal DNA palindrome of Tetrahymena pigmentosa has been determined. The sequence data show that 26 nucleotides at the very center are not palindromic. A segment of 34 base-pairs just outside the non-palindromic region is highly conserved between Tetrahymena pigmentosa and Tetrahymena thermophila, while the rest of the central regions show little sequence homology.     

Highly reiterated non-coding sequence in the genome of Plasmodium falciparum is composed of 21 base-pair tandem repeats

Clones containing highly reiterated DNA sequences were isolated from a Plasmodium falciparum genomic library. One clone, Rep2, was selected for further characterization by nucleotide sequence analysis. The results revealed that the insert of this clone is composed of tandemly arranged 21 base-pair imperfect repeats. These repeats are estimated to comprise approximately 1% of the P. falciparum genome and there are 10(4) to 2 X 10(5) copies, depending on the genome size estimate used for calculation. Moreover, the repeats are organized in clusters and do not appear to be transcribed in non-synchronized P. falciparum cultures.     

Restriction and modification enzymes detect no allosteric changes in DNA with bound lac repressor or RNA polymerase

A 203 base-pair fragment containing the lac operator/promoter region of Escherichia coli was inserted into the EcoRI site of the plasmid vector pKC7. Rates of restriction endonuclease cleavage of the flanking EcoRI sites and of several other restriction sites on the DNA molecule were then compared in the presence and absence of bound RNA polymerase or lac repressor. The rates were identical whether or not protein had been bound, even for sites as close as 40 base-pairs from a protein binding site. No difference was detected using supercoiled, nicked circular, or linear DNA substrates. No apparent change in the rates of methylation of EcoRI sites by EcoRI methylase was produced by binding the regulatory proteins.     

Organization of African green monkey DNA at junctions between alpha-satellite and other DNA sequences

Segments of African green monkey DNA containing sequences of the highly reiterated cryptic satellite DNA called α-satellite were selected from a library in λ bacteriophage. This λ library was constructed to enrich for monkey segments that contain (1) irregular regions of α-satellite and (2) α-satellite linked to other monkey sequences. At least 11 of 15 cloned monkey segments between 13 × 10³ and 16 × 10³ base-pairs in length, selected by hybridization to α-satellite, also include other monkey sequences.In general, α-satellite sequences close to the junctions with non-α-satellite DNA contain an abundance of divergent forms compared to the average frequency of such forms within total α-satellite. Many of the cloned segments are missing some of the HinIII sites that occur once in most monomer units of α-satellite, and likewise several of the cloned segments contain restriction sites that rarely occur in α-satellite as a whole. In some segments HinIII sites occur that are spaced at distances other than the basic multiple of 172 base-pairs. At least one of the cloned segments, however, is composed mainly of typical 172 base-pair long α-satellite monomer units.Several of these cloned DNAs have been mapped by restriction endonuclease digestion and Southern blot analysis and the arrangements of α-satellite and non-α-satellite sequences have been determined. In addition to segments that contain a boundary where satellite meets other types of sequence, some contain two such boundaries and thus satellite flanks a non-α-satellite segment. Further, two different types of non-α-satellite sequence appear to be common to more than one phage, perhaps indicating some recurring organization at boundaries.     

Extrachromosomal DNA forms of copia-like transposable elements, F elements and middle repetitive DNA sequences in Drosophila melanogaster. Variation in cultured cells and embryos

Drosophila melanogaster embryos and cells in culture were screened for the presence of unintegrated covalently closed circular DNA forms that hybridize to copia-like transposable elements, the F element and uncharacterized dispersed middle repetitive DNA elements. Our results indicate that the majority of copia-like elements (including copia, 297, 412, mdg1, mdg3 and gypsy), the F elements, and 9 of 12 middle repetitive DNA elements are present as free DNA forms in cultured cells and embryos. An 18 base-pair inverted repeat has been reported to flank the long direct repeat of mdg3, implying that mdg3 is not an orthodox copia-like element; however, we have sequenced two independently isolated mdg3 clones and shown that the inverted repeat is not part of the element. The relative abundance with which free DNA forms are found varies between the cultured cells used, and between cultured cells and embryos. This variation, which can be up to 20-fold for some elements, does not correlate well with either the amount of element-specific poly(A)+ RNA present per cell or the number of element-specific sequences integrated in the genome.     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Glucosylated nucleotide sequences from T-even bacteriophage deoxyribonucleic acids

1. The frequencies of various pyrimidine nucleotide sequences in phage-T2VG111 DNA and phage-T6 DNA have been measured. The hydroxymethylcytosine nucleotides that bear glucosyl groups in phage-T2VG111 DNA and gentiobiosyl groups in phage-T6 DNA are not randomly distributed. 2. Sequences in which two or three hydroxymethylcytosine nucleotides are terminated at both ends by purine nucleotides bear only one sugar substituent and this is attached to the first hydroxymethylcytosine when the sequences are written in the conventional notation; i.e. with the 5′-phosphate before and the 3′-phosphate after each nucleoside residue. This resembles the distribution of glucosyl residues previously found in the corresponding sequences from phage-T2 DNA. Sequences in which one hydroxymethylcytosine nucleotide is attached through the 3′-position to a purine nucleotide are more fully glucosylated in phage-T2VG111 DNA and phage-T6 DNA than in the DNA of phage T2. 3. Pyrimidine sequences from the DNA of phage T6(S) have been found to contain glucosyl but not gentiobiosyl residues. The relative amounts of the sequences isolated indicate that the distribution of the glucosyl residues in this DNA resembles that of the gentiobiosyl groups in phage-T6 DNA. 4. It is suggested that the distributions of glycosyl substituents in these T-even-phage deoxyribonucleic acids reflect the specificity requirements of the α-glucosyltransferases induced by the different phages. The results obtained with the DNA of phage T6(S) suggest that this phage induces the usual phage-T6 α-glucosyltransferase but not the phage-T6 β-glucosyltransferase.