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R B Hallick

Publications and source records attributed to R B Hallick.

At least 37 records · Page 2Linked to original sources
PubMed1992

Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites.

The chloroplast genes of Euglena gracilis contain more than 60 group II and 47 group III introns. Some Euglena chloroplast genes also contain twintrons, introns-within-introns. Two types of twintrons have previously been described, a group II twintron and a mixed group II/group III twintron. We report that four introns, three within the RNA polymerase subunit gene rpoC1 and one within ribosomal protein gene rpl16, with mean lengths twice typical group III introns, are a new type of twintron. The group III twintrons are composed of group III introns within other group III introns. The splicing of the twintrons was analyzed by PCR amplification, cloning and sequencing of cDNAs, and Northern hybridization. Excision of each group III twintron occurs by a two-step, sequential splicing pathway. Removal of the internal introns precedes excision of the external introns. Splicing of internal introns in three of the four group III twintrons involves multiple 5'- and/or 3'-splice sites. With two of the twintrons the proximal 5'-splice site can be spliced to an internal 3'-splice site, yielding alternative 'pseudo' fully spliced mRNAs. Excised group III introns of the rpl16 twintron are not linear RNA molecules but either lariat or circular RNAs, probably a lariat. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.

Alternative Splicing↗
PubMed1992

Chloroplasts.

New features of chloroplast gene expression are continually being discovered, particularly in the area of post-transcriptional RNA processing. RNA editing of chloroplast pre-mRNAs occurs in both monocotyledons and dicotyledons, and involves both initiator and internal codons. The view of introns as mobile genetic elements expands both with the identification of additional twintrons in Euglena chloroplast genes and with studies on the homing group I introns of Chlamydomonas.

Chloroplasts↗
PubMed1992

Simple Determination of the CO(2)/O(2) Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [C]Glycerate 3-Phosphate.

A new method is presented for measurement of the CO(2)/O(2) specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [(14)C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. (14)CO(2) fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO(2) in O(2)-saturated water and carboxylase only with 160 micromolar CO(2) under N(2). Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [(14)C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 +/- 4), from the green alga Chlamydomonas reinhardtii (66 +/- 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.

Journal Article↗
PubMed1991

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.

Amino Acid Sequence↗
PubMed1991

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

The psbF gene of chloroplast DNAs encodes the beta-subunit of cytochrome b-559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre-mRNA, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term 'twintron' to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.

Animals↗
PubMed1991

Intercistronic group III introns in polycistronic ribosomal protein operons of chloroplasts.

A novel ribosomal protein operon in the Euglena gracilis chloroplast genome was characterized. It encodes the genes for ribosomal proteins S4 and S11 (rps4 and rps11). The coding region of the rps11 gene is interrupted by two introns of 107 and 100 bp. The introns belong to a distinct class known as group III introns. The major transcript from this operon was characterized as a fully spliced dicistronic rps4-rps11 mRNA by RNA blot analysis, primer extension sequencing, and cDNA cloning and sequencing. An additional 95 nucleotide (nt) group III intron was identified in the 123 nt rps4-rps11 intercistronic region. The identification of the intercistronic intron between the rps4 and rps11 genes was unexpected. Other RNA transcripts from regions of the genome that could potentially contain intercistronic introns were re-examined and two other intercistronic, group III introns were found. These are located in a large ribosomal protein operon between the genes for the ribosomal proteins L23 and L2, and between L14 and L5. There are at least 50 group III introns in the E. gracilis chloroplast genome. All but 6 are found in genes encoding protein components of the transcriptional and translational apparatus. The distribution of group III introns and the unusual location of intercistronic group III introns may reflect some aspect of gene expression, or provide some insight into the mechanism of their splicing.

Amino Acid Sequence↗
PubMed1990

The Euglena gracilis chloroplast rpoB gene. Novel gene organization and transcription of the RNA polymerase subunit operon.

The rpoB gene coding for a beta-like subunit of the chloroplast DNA-dependent RNA polymerase has been located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. We have determined 5760 base-pairs of DNA sequence, including 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resembles the E. coli rpoB-rpoC and higher plant chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with a predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp respectively and a Group II intron of 309 bp. All other known rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNA, as well as rpoB-rpoC1 and rpoB1-rpoC1-rpoC2 mRNAs were identified.

Amino Acid Sequence↗
PubMed1990

Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases.

Animals↗
PubMed1989

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.

Amino Acid Sequence↗
PubMed1988

Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.

The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities. We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE). Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes. The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies. The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis. In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259: 14880-14887). From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

Amino Acid Sequence↗
PubMed1988

The two genes for the P700 chlorophyll a apoproteins on the Euglena gracilis chloroplast genome contain multiple introns.

We have determined the complete nucleotide sequence of the two genes encoding the P700 chlorophyll a apoproteins of the photosystem I reaction center of the Euglena gracilis chloroplast genome. The two genes are separated by 77 bp, are of the same polarity, and span a region which is greater than 9.0 kbp. The psaA gene (751 codons) is interrupted by three introns and the psaB gene (734 codons) by six introns. The introns range in size from 361 to 590 bp, whereas the exons range in size from 42 to 1,194 bp. The introns are extremely AT rich with a pronounced base bias of T greater than A greater than G greater than C in the RNA-like strand. Like other interrupted protein genes in the Euglena chloroplast genome, the psaA and psaB introns are similar to mitochondrial group II introns in having the splice junction consensus sequence, 5' GTGCGNTTCG ..... INTRON ..... TTAATTTTAT 3' and conserved secondary structural features. Except for the placement of the first intron, the intron-exon organization of these two highly homologous genes is not conserved. The other introns fall at or near putative surface domains of the predicted gene products. The psaA and psaB gene products are 74% homologous to one another and 93% and 95% homologous, respectively, to the psaA and psaB gene products of higher plant chloroplasts. The predicted secondary structure derived from the primary amino acid sequence has 11 potential membrane-spanning domains.

Amino Acid Sequence↗
PubMed1988

Organization of the psbE, psbF, orf38, and orf42 gene loci on the Euglena gracilis chloroplast genome.

The genes for cytochrome b559, designated psbE and psbF, and two highly conserved open reading frames of 38 and 42 codons have been located and characterized on the chloroplast genome of Euglena gracilis. The organization of the genes is psbE - 8 bp spacer - psbF - 110 bp spacer - orf38 - 87 bp spacer - orf42. All genes are of the same polarity. The psbE gene contains two introns of 350 and 326 bp. The psbF gene contains a single large intron of 1,042 bp. The orf38 and orf42 loci lack introns. The introns are extremely AT rich with a pronounced base composition bias of T greater than A greater than G greater than C in the mRNA-like strand and group II-like boundary sequences at their 3' and 5' ends having the consensus 5'-GTGTG .. INTRON .. TTAATTTNAT-3'. The psbE gene consists of 82 codons and encodes a polypeptide with a predicted molecular weight of 9,212. The psbF gene consists of 42 codons, which specify a polypeptide with a predicted molecular weight of 4,785. The highly conserved open reading frames of 38 and 42 codons code for polypeptides with predicted molecular weights of 4,405 and 4,426, respectively. The gene products of psbE, psbF, orf38 and orf42 are, respectively, 69.5%, 70% and 61.5% identical to those found in higher plants. The predicted secondary structure of the proteins from hydropathy plots is consistent with each containing a single membrane-spanning domain of at least 20 amino acids. Each of the genes is preceded by sequences which may serve as ribosome binding sites. All four genes are transcribed.

Amino Acid Sequence↗
PubMed1988

Organization of ribosomal protein genes rpl23, rpl2, rps19, rpl22 and rps3 on the Euglena gracilis chloroplast genome.

The nucleotide sequence (4,814 bp) was determined for a cluster of five ribosomal protein genes and their DNA flanking regions from the chloroplast genome of Euglena gracilis. The genes are organized as rpl23-150 bp spacer-rpl2-59 bp spacer-rps19-110 bp spacer-rpl22-630 bp spacer-rps3. The genes are all of the same polarity and reside 148 bp downstream from an operon for two genes of photosystem I and four genes of photosystem II. The Euglena ribosomal protein gene cluster resembles the S-10 ribosomal protein operon of Escherichia coli in gene organization and follows the exact linear order of the analogous genes in the tobacco and liverwort chloroplast genomes. The number and positions of introns in the Euglena ribosomal protein loci are different from their higher plant counterparts. The Euglena rpl23, rps19 and rps3 loci are unique in that they contain three, two and two introns, respectively, whereas rpl2 and rpl22 lack introns. The introns found in rpl23 (106, 99 and 103 bp), rps19 (103 and 97 bp) and rps3 intron 2 (102 bp) appear to represent either a new class of chloroplast intron found only in constitutively expressed genes, or possibly a degenerate version of Euglena chloroplast group II introns. They are deficient in bases C and G and extremely rich in base T, with a base composition of 53-76% T, 25-34% A, 3-10% G and 2-7% C in the mRNA-like strand. These six introns show minimal resemblance to group II chloroplast introns. They have a degenerate version of the group II intron conserved boundary sequences at their 5' and 3' ends. No conserved internal secondary structures are apparent. By contrast, rps3 intron 1 (409 bp) has a potential group II core secondary structure. The five genes, rpl23 (101 codons), rpl2 (278 codons), rps19 (95 codons), rpl22 (114 codons) and rps3 (220 codons) encode lysine-rich polypeptides with predicted molecular weights of 12,152, 31,029, 10,880, 12,819, and 25,238, respectively. The Euglena gene products are 18-50%, and 29-58% identical in primary structure to their E. coli and higher plant counterparts, respectively. Oligonucleotide sequences corresponding to Euglena chloroplast ribosome binding sites are not apparent in the intergenic regions. Inverted repeat sequences are found in the upstream flanking region of rpl23 and downstream from rps3.

Amino Acid Sequence↗
PubMed1988

Trans-splicing in chloroplasts: the rps 12 loci of Nicotiana tabacum.

The rps12 gene in tobacco chloroplasts consists of three exons that code for a polypeptide with homology to Escherichia coli ribosomal protein S12. C-terminal exons 2 and 3 of rps12 are located in the inverted repeat regions of the tobacco chloroplast genome. Exon 1 of rps12 is 29 kilobase pairs downstream of the nearest copy of exons 2 and 3 and 69 kilobase pairs away from the distal copy of exons 2 and 3. RNA gel blot hybridization analysis and primer extension sequencing of cDNA to rps12 encoding RNAs indicate that exon 1 and exons 2 and 3 are encoded on separate transcripts. Exon 1 and exons 2 and 3 are covalently ligated in the correct reading frame in rps12 mRNA. These results indicate that a bimolecular (trans-) splicing event occurs during the formation of mature rps12 mRNA.

Chloroplasts↗