Histone genes of the sea urchin (S. purpuratus) cloned in E. coli: Order,polarity, and strandedness of the five histone-coding and spacer regions

Sea urchin (S. purpuratus) histone DNA of constructed plasmid chimeras cloned in E. coli was cleaved with the restriction endonucleases Eco RI, Hind III, Sal I, Bam I, and Hha I. The resulting fragments were ordered and isolated directly from agarose gels or cloned into other plasmids. Each fragment hybridized to one or another of the five histone mRNAs and elucidated the order of the histone genes in each of the cloned fragments. Some DNA did not hybridize to histone mRNAs and was identified as spacer DNA located between coding regions.Total sea urchin DNA was cleaved with restriction endonucleases, fractionated on agarose gels, and hybridized to histone mRNAs or histone DNA. The results revealed the order of the five histone genes in the histone gene repeat unit and demonstrate that the histone spacer DNAs have little sequence homology to other genes. Exonuclease III digestion of specific linear chimeric histone DNA plasmids followed by hybridization with mRNAs demonstrated the existence of all five histone genes on one strand of DNA and the 5′-3′ polarity of that strand. These results, in conjunction with the data of Wu et al. (1976), allow us to construct a map of coding and spacer sequences in the transcribed strand of the S. purpuratus histone gene repeat unit:

     

Nucleotide Sequence Homology Exists between the Chloroplast and Nuclear Ribosomal DNAs of Euglena gracilis

The nuclear and chloroplast ribosomal DNAs from Euglena were shown to have specific regions of nucleotide sequence homology. The regions of homology were identified by hybridization of restriction endonuclease DNA fragments of cloned chloroplast and nuclear ribosomal DNAs to one another. The regions of homology between these two ribosomal DNAs were in that part of the genes that code for the 3′ end of the small rRNAs (16S and 19S) and near or at the DNA sequences coding for the 5S RNAs. The nucleotide sequence homology between these regions was estimated to be approximately 94% by the melting point depression of a hybrid formed between the two ribosomal DNAs.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol?γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol?γ from yeast to human.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

Cloning and structural analysis of the snap-back DNA of Pharbitis nil

We isolated and cloned DNA fragments that exist as inverted-repeat structures in the genome of Pharbitis nil. The method used exploited the fact that if inverted repeat DNA is present in the DNA fragment, intramolecular double-stranded structures can be partly formed within single-stranded DNA molecules after denaturation and rapid renaturation of the fragment. The rapidly renaturing DNA fragments (termed snap-back DNA) were isolated by hybroxylapatite column chromatography and treatment with mungbean nuclease and were cloned into the pUC9 vector. Four snap-back DNA members out of thousands of independent clones obtained were characterized with respect to the reiteration frequency and the nucleotide sequences. When used as probes in Southern hybridization experiments, some of the members identified restriction fragment length polymorphism among the cultivars, suggesting that these sequences might be fluid in the genome. One of the four clones has regions of nucleotide sequence homology to those of inverted-repeat regions in the transposon Taml of Antirrhinum majus.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.