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R B Hallick

Publications and source records attributed to R B Hallick.

At least 73 records · Page 4Linked to original sources
PubMed1982

Synthetic deoxyoligonucleotides as general probes for chloroplast transfer RNA genes.

The utility of chemically synthesized deoxyoligonucleotides as hybridization probes for the detection of tRNA genes has been examined. Chloroplast tRNA genes were chosen for this study. Deoxyoligonucleotides complementary to highly conserved regions of chloroplast tRNA genes of both higher plants and Euglena gracilis were chemically synthesized. These synthetic probes have been used to detect tRNA genes by Southern hybridizations to restriction fragments of chloroplast DNAs. This new method of tRNA gene mapping and the oligonucleotides synthesized may be of general application to many chloroplast genomes. This is illustrated by the detection of known and unknown tRNA genes of Euglena gracilis and spinach, and unknown tRNA genes of maize and cucumber chloroplast DNAs. The precise locus and polarity of the Euglena gracilis chloroplast tRNAPhe gene has been determined. We also describe experiments which relate to the effects of the time of hybridization, the stringency of washing, and of base pair mismatches on the hybridization signal.

Chloroplasts↗
PubMed1982

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNATyr-tRNAHis-tRNAMet-tRNATrp-tRNAGlu-tRNAGly gene cluster.

A tRNA coding locus in the Bam-Sal 9 region of Euglena gracilis Pringsheim strain Z chloroplast DNA was chosen for detailed study. This DNA contains the previously mapped tRNA coding sequences of the adjacent Euglena chloroplast EcoRI products of EcoV and EcoH (Orozco, E. M., Jr., and Hallick, R. B. (1982) J. Biol. Chem. 257, 3258-3264). The 3.2-kilobase pair Bam-Sal 9 fragment was cloned into the BamHI and SalI cut plasmid vector pBR322, resulting in the recombined plasmid pPG76. The tRNA coding locus was mapped to a region of Bam-Sal 9 that contains portions of both EcoH and EcoV. The DNA sequence of 1-kilobase pair Bam-Sal 9, containing the entire tRNA coding locus, was determined. A cluster of six tRNA genes was found. The gene organization is as follows, where bp is base pair: tRNATyrGUA-64 bp spacer-tRNAHisGUG-14 bp spacer-tRNAMetCAU-4 bp spacer-tRNATrpCCA-27 bp spacer-tRNAGluUUC-6 bp spacer-tRNAGlyUCC. The tRNAMetCAU is believed to be an elongator tRNA. The first four genes are within EcoV. The EcoRI cleavage site that separates EcoV and EcoH is in the tRNAGlu gene. The tRNAGly gene is in EcoH. This is the largest known chloroplast tRNA gene cluster.

Amino Acids↗
PubMed1982

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence.

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.

Base Sequence↗
PubMed1982

Euglena gracilis chloroplast transfer RNA transcription units. I. Physical map of the transfer RNA gene loci.

The locations of transfer RNA genes with respect to the restriction endonuclease cleavage map of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. Purified chloroplast tRNAs were treated with snake venom phosphodiesterase to remove the 3'-CCA terminus, and radioactively labeled by the action of Escherichia coli tRNA nucleotidyltransferase in the presence of [alpha-32P]CTP. Chloroplast DNA was treated individually and with combinations of the enzymes Bal I, Bam HI, Eco RI, Pst I, Pvu II, Sal I, and Xho I. The location of tRNA genes with respect to the cleavage sites for these enzymes was determined by hybridization of the 32P-labeled tRNAs to membrane filter blots of the chloroplast DNA restriction nuclease fragments following gel electrophoresis. The 145-kilobase pair genome was resolved into nine areas of strong tRNA hybridization, separated by areas of weak or no tRNA hybridization. The loci of tRNA genes are within the Eco RI fragments Eco A, B, G, H, I, J', P, Q, and V.

Base Sequence↗
PubMed1982

Euglena gracilis chloroplast transfer RNA transcription units. II. Nucleotide sequence analysis of a tRNAVal-tRNAAsn-tRNAArg-tRNALeu gene cluster.

The tRNA-coding locus of the 8.2-kilobase pair (kbp) Eco RI fragments Eco G of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA was chosen for detailed analysis. Two recombinant plasmids, pPG14, containing Eco G and the vector pMB9, and pEZC23, containing the chloroplast DNA fragment HindIII B cloned in pBR322 were employed for the study. The tRNA locus was mapped to an 0.8-kbp region of Eco G also present in HindIII B. The DNA sequence of a 1.6 kbp from HindIII B, containing the entire tRNA gene locus was determined. Four tRNA genes were identified from the DNA sequence. The gene organization is tRNAVal-16 bp spacer-tRNAAsn-3 bp spacer-tRNAArg-45 bp spacer-tRNALeu. The tRNALeu gene is of the opposite polarity as the other three genes. This is the first evidence of such a tRNA cluster for a chloroplast genome. Also evident from the DNA sequence, 132 bp from the 5'-end of the tRNALeu gene, is a putative gene or pseudogene for a chloroplast protein.

Base Composition↗
PubMed1982

Transcription of E. coli and Euglena chloroplast tRNA gene clusters and processing of polycistronic transcripts in a HeLa cell-free system.

The transcription of cloned tRNA genes that are clustered in the Escherichia coli and Euglena gracilis chloroplast genome was studied in a HeLa cell-free extract. RNA polymerase III transcribes the tRNA gene clusters into polycistronic primary transcripts, consisting of pre-tRNAVal-tRNAAsn-tRNAArg and pre-tRNAAsp-tRNATrp transcribed from the Euglena chloroplast and E. coli tRNA locus, respectively. A UV5-lac promoter in the 5'-flanking sequence of the E. coli tRNA gene cluster can be removed without effect on transcription initiation frequency. During transcription of the tRNA gene clusters, RNA polymerase III initiates predominantly, if not solely, with the first tRNA gene. Upon removal of the first tRNA gene, RNA polymerase III initiates transcription with the following tRNA gene. The primary transcripts are subsequently processed to mature tRNAs. RNA fingerprint analysis and reincubation experiments were used to establish the steps of tRNA maturation of the pre-tRNAVal-tRNAAsn-tRNAArg. The 3' trailer is processed prior to the removal of 5' leader and before cleavage of the primary transcript in the spacer sequences. The pre-tRNAs are then processed to mature tRNA molecules.

Cell-Free System↗
PubMed1980

Euglena gracilis chloroplast ribosomal RNA transcription units. I. The location of transfer RNA, 5 S, 16 S, and 23 S ribosomal RNA genes.

The organization of the genes in the rRNA coding region of Euglene gracilis Pringsheim strain Z chloroplast DNA has been determined. 125I labeled 16 S rRNA, 125I labeled 23 S rRNA, and [32P]tRNAs were hybridized to restriction nuclease fragments from cloned chloroplast DNA segments. The coding regions for 16 S and 23 S rRNAs have been located to within 1.75 and 3.1 kbp, respectively. Transfer rRNA genes are located in the 16 S to 23 S rRNA spacer region. Ribosomal RNAs and tRNAs are encoded on the same DNA strand in the 6.2 kbp tandemly repeated rRNA gene region. The order of genes, which is repeated three times in chloroplast DNA, is 16 S rRNA--tRNAS--23 S rRNA--5 S rRNA. Each set of genes is clustered in a region of 5.0 kbp. The organization and size of the genes is as expected if they are all part of a single transcription unit.

Chloroplasts↗
PubMed1980

Euglena gracilis chloroplast ribosomal RNA transcription units. II. Nucleotide sequence homology between the 16 S--23 S ribosomal RNA spacer and the 16 S ribosomal RNA leader regions.

The DNA sequences of two segments of the ribosomal RNA transcription units of Euglena gracilis Pringsheim strain Z chloroplast DNA have been determined. The first is from the 16 S to 23 S rRNA spacer region. The nucleotide sequence determined includes 64 bp from the 3'-end of the 16 S rRNA gene, the adjacent 87-bp spacer containing 68A-T base pairs, a tRNAIle gene, a 9-bp spacer, a tRNAAla gene, a spacer of approximately 15 bp, and the first 120 bp from the 5'-end of the 23 S rRNA gene. The gene organization of the 16 S to 23 S rRNA spacer, the identity of the tRNA genes, and the tRNA anticodons for the E. gracilis rRNA transcription units are identical with that of the rrnA, D, and X operons of Escherichia coli. The second DNA segment which was sequenced is from a region preceding the 5'-end of the 16 S rRNA gene. Within a continuous region of 189 bp in this 16 S rRNA leader sequence, 68% of the bases are homologous to the 16 S rRNA to 23 S rRNA spacer region. This homology includes the 3'-end of the 16 S rRNA gene, the adjacent spacer, and a complete "pseudo" tRNAIle gene. This leader sequence which has the same polarity as the rRNA transcripts, is flanked by nucleotide sequences resembling partial tRNA genes.

Base Sequence↗
PubMed1980

Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome.

The specificity of transcription of Euglena gracilis Z chloroplast DNA by chloroplast DNA-dependent RNA polymerase in a transcriptionally active chromosome (Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045) has been studied. RNA molecules are both initiated and elongated in vitro. The RNA transcripts have been characterized as to their size, nuclease sensitivity, 5'-terminal oligonucleotides, and coding locus on the chloroplast genome. RNA labeled in vitro at the 5' end with [gamma-32P]ATP was digested with RNase T1, RNase A, and S1 nuclease. The resulting 5'-gamma-32P-oligonucleotides were fractionated by gel electrophoresis. In each case, one or two discrete products were obtained, consistent with initiation in vitro only at defined loci. RNA labeled in vitro with [alpha-32P]ATP or CTP has been hybridized to Southern (Southern, E.M. (1975) J. Mol. Biol. 98, 503-517) transfers of restriction endonuclease fragments of chloroplast DNA. The most abundant in vitro transcripts hybridize to chloroplast DNA fragments coding for 23 S, 16 S, and 5 S rRNAs. Only the coding strands of the rRNA genes are transcribed. Non-rDNA sequences of chloroplast DNA are also selectively transcribed but at much lower levels. The transcriptionally active chromosome has proved to be an ideal biochemical preparation for the study of selective transcription of cell organelle DNA.

Chloroplasts↗
PubMed1979

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.

Chloroplasts↗
PubMed1979

Base composition heterogeneity of Euglena gracilis chloroplast DNA.

Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content.

Animals↗
PubMed1979

Transcription program of the chloroplast genome of Euglena gracilis during chloroplast development.

RNA transcription in Euglena gracilis chloroplasts has been characterized by hybridization of RNA from cells at different stages of chloroplast development to (3)H-labeled chloroplast DNA restriction endonuclease fragments. Chloroplast DNA was digested into five fragments of 53,000, 35,000, 25,000, 10,000, and 6900 base pairs with Pst I. The 53,000-base-pair DNA was further cleaved by BamHI digestion. Eight different DNA fragments, of known restriction nuclease map location and accounting for the entire genome, were labeled in vitro by means of the nick translation reaction of DNA polymerase I with [(3)H]dTTP as a substrate. RNA was isolated from dark-adapted Euglena cells, and from cells at an early (4 hr) and a late (72 hr) stage of light-induced chloroplast development. The RNAs were hybridized in solution to each (3)H-labeled chloroplast DNA fragment. From the extents and kinetics of the reactions, a temporal program for RNA transcription from defined regions of the chloroplast genome could be described. Different classes of transcription units are present, including RNAs (i) continuously present throughout development, (ii) induced at the onset of development, (iii) repressed early in development, and (iv) induced late in development.

Journal Article↗
PubMed1978

Mapping of transcribed regions of Euglena gracilis chloroplast DNA.

RNA transcription from defined regions of the Euglena gracilis chloroplast genome has been characterized by hybridization of total cell RNA to 3H-labeled chloroplast DNA restriction endonuclease fragments. Chloroplast DNA was digested into five fragments of 53, 35, 25, 10, and 6.9 kilobase pairs (kbp) with Pst1. The 53-kbp DNA was also subfractionated by BamHI digestion. The extent of transcription of the Pst1 fragments was found to be 30, 17, 15, 2.2, and 2.3 kb of RNA, respectively. The total amount of RNA transcription of 67 kb represents 26% to the double-strand information content of the genome. Transcribed regions are dispersed throughout the DNA. The RNA transcripts are present in two major abundance classes in the cell. High abundance transcripts of approximately 10(6) copies/cell were mapped in the rRNA gene region of the 53-kbp fragment and in the 35-kbp fragment. Low abundance transcripts of approximately 1000--4000 copies/cell were mapped in all five Pst fragments.

Animals↗
PubMed1977

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.

Animals↗
PubMed1977

Expression of the chloroplast ribosomal RNA genes of Euglena gracilis during chloroplast development.

The cellular content and transcription program of the chloroplast ribosomal RNA genes of Euglena gracilis Z have been determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ([3H]ctrDNA). Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E. coli DNA polymerase I with [3H]TTP as a substrate. The [3H] DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA. The rRNA-[3H]ct rCNA hybrid was isolated by chromatography on hydroxylapatite. The [3H]ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of [3H]DNA-DNA and [3H]DNA-RNA hybrids. [3H]ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0,4,8,12,24,48, and 72 h after the onset of chloroplast development. From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made. Chloroplast rRNA increases from 2 to 26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases two- to threefold. Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than twofold, while the number of copies of rRNA per cell increases sixfold. The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during developmental.

Animals↗
PubMed1977

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.

Animals↗
PubMed1977

Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation.

Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases. ATA has been shown to inhibit the following enzymes in vitro: DNAse I, RNAse A, S1 nuclease, exonuclease III, and restriction endonucleases Sal I, Bam HI, Pst I and Sma I. The observed inhibition is consistent with the proposal by Blumenthal and Landers (BBRC 55, 680, 1973) that most nucleic acid binding proteins will be sensitive to ATA. The action of ATA as a nuclease inhibitor can be used to advantage in the isolation of cellular nucleic acids.

Aurintricarboxylic Acid↗