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L Bogorad

Publications and source records attributed to L Bogorad.

At least 37 records · Page 2Linked to original sources
PubMed1993

5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.

We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon illumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.

Animals↗
PubMed1993

Co-transcription pattern of an introgressed operon in the maize chloroplast genome comprising four ATP synthase subunit genes and the ribosomal rps2.

Several examples of the introduction of a gene from one gene complex into another (introgression) are found when chloroplast RP gene clusters are compared to those in Escherichia coli or cyanobacteria. Here we describe the transcript pattern of one such cluster from maize (Zea mays) that includes the genes for 4 subunits of the thylakoid ATP synthase (atpI, H, F, A) and the rps2 gene. Twelve transcript species covering the size range from 7,000 to 800 nt were identified in RNA isolated from dark-grown and greening maize seedlings, and several of them were characterized by reverse transcription analysis. A major species of 6,200 nt, with its 5' end at 181 nt upstream of the initiating ATG of rps2, contained the transcripts of all the 5 genes. Two further sets of transcripts having their 5' ends ca. 120 and 50 nt upstream of the initiation codons of the atpI and atpH genes were also identified. Thus, this plastid gene cluster in maize is functionally organized as an operon with additional regulatory features to allow for increased accumulation of mRNAs for the thylakoid components.

Adenosine Triphosphatases↗
PubMed1993

Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes.

Possible roles of untranslated sequences at the 3' ends of chloroplast genes, which include inverted repeat elements, were investigated in Chlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or psaB 3' flanking regions were coupled in various arrangements 3' to a chimeric gene consisting of a Chlamydomonas chloroplast atpB promoter sequence fused 5' to the Escherichia coli uidA (GUS) structural gene. These genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojectile bombardment. Transformants harboring chimeric GUS genes fused to rbcL or psaB gene 3' inverted repeat sequences in their normal forward orientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harboring the same gene lacking an inverted repeat sequence at its 3' end. Thus, the 3' flanking regions of the rbcL and psaB genes can define the location of the 3' terminus of a transcript in vivo. In chloroplast transformants harboring chimeric GUS genes fused to multiple inverted repeat sequences in their normal forward orientations, only GUS transcripts accumulated that were terminated by the first inverted repeat sequence. The latter data suggest that the 3' ends of these RNAs are the products of either transcription termination or endonucleolytic cleavage. Analyses of GUS transcripts in transformants harboring GUS genes terminated by rbcL or psaB gene 3' flanking regions in reversed orientations indicate that transcript 3' end formation in vivo requires nucleotide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3' inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamydomonas chloroplast transcripts studied.

Animals↗
PubMed1993

Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation.

Changes in the relative sizes of pools of transcripts of organelle genes during plastid development are common in flowering plants, but technical difficulties have prevented direct determinations of the effects of changes in rates of transcription and degradation on such fluctuations. It has been possible to follow both rates in Chlamydomonas reinhardtii. In synchronous or asynchronous cultures of cells grown in 12 h light/12 h dark periods, sizes of pools of transcripts of the chloroplast genes atpA, atpB, tufA, and psaB fluctuate. Differences in chloroplast transcript abundances in light/dark cycles were found to be cell cycle-independent but controlled by either an endogenous rhythm (atpA, atpB, and tufA) or by light (psaB). In vivo labeling experiments showed that transcriptional regulation and light/dark-regulated degradation both contribute, in gene-specific manners, to the level of transcripts of individual C. reinhardtii chloroplast genes in cells grown in alternating light/dark cycles.

Animals↗
PubMed1992

Excitation energy transfer from phycocyanin to chlorophyll in an apcA-defective mutant of Synechocystis sp. PCC 6803.

A greenish mutant of the normally blue-green cyanobacterium Synechocystis sp. PCC 6803, designated UV6p, has been isolated and characterized. UV6p possesses functional photosystems I and II (PSI and PSII) but lacks normal light harvesting phycobilisomes because allophycocyanin is absent and core-specific linker proteins are almost entirely absent. The mutation responsible for the UV6p phenotype has been identified; it is a base substitution which results in the creation of a termination codon within the coding region of the apcA gene. Phycocyanin (PC) and phycobilisome rod linker proteins are present in UV6p and, despite the absence of core components, at least 35% of the PC is associated with rod linker proteins. At 77 K, light absorbed by PC of UV6p elicits PSI fluorescence comparable to that of wild type cells but produces greatly diminished PSII fluorescence. The results indicate that the assembly of rods is independent of cores and that light energy absorbed by rods can be transferred principally and directly to PSI. This energy transfer pathway, which may also be present in wild type, may have a regulatory role in maintaining the balance of input of excitation energy into PSI versus PSII during photosynthesis.

Amino Acid Sequence↗
PubMed1992

Two types of chloroplast gene promoters in Chlamydomonas reinhardtii.

Structures of the promoters of Chlamydomonas reinhardtii plastid atpB and 16S rRNA-encoding genes were analyzed in vivo. Chimeric constructs, containing the Chlamydomonas chloroplast atpB or 16S rRNA-encoding gene promoter coupled to the Escherichia coli uidA (beta-glucuronidase, GUS) reporter gene and bordered by C. reinhardtii chloroplast sequences, were stably introduced into the chloroplast of Chlamydomonas by microprojectile bombardment. Activity of the promoters in the chloroplast of GUS gene-positive transformants was assayed by measuring the abundance of GUS transcripts and determining the relative rates of GUS transcription in vivo. Deletion analyses of the 16S rRNA gene and atpB promoter fragments showed that the two promoters differ structurally. The 16S rRNA gene promoter resembles the bacterial sigma 70 type with typical -10 and -35 elements. The atpB promoter, on the other hand, lacks a conserved motif in the -35 region but contains, in the -10 region, a characteristic octameric palindrome (TATAATAT) that is conserved in the promoter sequences of some other C. reinhardtii chloroplast genes. For maximum activity, the atpB promoter requires sequences of approximately 22 base pairs upstream and approximately 60 base pairs downstream of the transcription start site.

Animals↗
PubMed1992

Transient expression from cab-m1 and rbcS-m3 promoter sequences is different in mesophyll and bundle sheath cells in maize leaves.

Cell-specific and light-regulated expression of the beta-glucuronidase (GUS) reporter gene from maize cab-m1 and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-m1 gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC). Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-m1 is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination of dark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-m1 region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-m1-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region of rbcS-m3 used here could be responsible for most of the light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC.

Base Sequence↗
PubMed1992

The cab-m7 gene: a light-inducible, mesophyll-specific gene of maize.

Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclear cab multigene family encoding the chlorophyll a- and b-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3' probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designated cab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. This cab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts of cab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of the cab-m7 gene resemble those found in the 5'-flanking regions of some other plant genes.

Amino Acid Sequence↗
PubMed1992

A rice cab gene promoter contains separate cis-acting elements that regulate expression in dicot and monocot plants.

The major light-harvesting chlorophyll a/b binding proteins of the photosynthetic apparatus are encoded by families of nuclear cab genes. The expression of most cab genes is tissue specific and photoregulated in angiosperms. In transgenic tobacco plants, expression of the reporter gene beta-glucuronidase (GUS) is photoregulated and tissue specific from 5' upstream sequences of the rice cab1R gene; deletion of sequences upstream from position -170 with respect to the transcription start site eliminates the enhanced and photoregulated expression in the transgenic plants. Using an in situ transient expression assay, we have determined that the sequence OCT-R, an octamer repeat that lies within the -269 to -170 region of cab1R, is essential for photoregulated expression of the chimeric GUS gene in leaf cells of maize and rice but is not required for expression in illuminated tobacco leaves. Conversely, box III*- and G-box-like sequences found near OCT-R in cab1R are necessary for high-level transient expression of the reporter gene in tobacco leaf tissue but are not required for transient expression in maize or rice leaves.

Base Sequence↗
PubMed1991

A residue substitution in phosphoribulokinase of Synechocystis PCC 6803 renders the mutant light-sensitive.

We have isolated a light-sensitive mutant (BRLS) of the photosynthetic cyanobacterium Synechocystis 6803 (S. 6803) that does not survive exposure to bright light: 70% of BRLS cells die upon exposure to light of greater than 3,000 lux for 2 h. A complementing DNA fragment from wild-type cells and the corresponding DNA from the BRLS cells have been cloned and sequenced. An open reading frame is found to encode phosphoribulokinase, a key enzyme in the enzyme system for photosynthetic carbon reduction (ES-PCR). The deduced peptide sequence of this enzyme is highly homologous to eukaryotic phosphoribulokinases but is not similar to known prokaryotic phosphoribulokinases. The mutation responsible for the phenotype of BRLS is a single nucleotide change that results in substitution of phenylalanine for Ser-222 in the phosphoribulokinase. The catalytic activity and the apparent affinity for ATP of the mutated kinase are about one-tenth and one-seventh those of the wild-type kinase, respectively. Furthermore, the mutated kinase is selectively degraded in BRLS cells in bright light. Degradation of the mutated kinase and cell death in bright light can be suppressed by inhibiting photosynthetic electron flow (PS-EF) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The data indicate that PS-EF is not impeded by an impaired ES-PCR although the ES-PCR activity is controlled by the rate of PS-EF. Continued PS-EF in the absence of the normal substrates for carbon reduction appears to result in damage to cellular components essential for life or in the generation of lethal components.

Amino Acid Sequence↗
PubMed1991

Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene.

The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation.

Amino Acid Sequence↗
PubMed1991

Deletion of the psbG1 gene of the cyanobacterium Synechocystis sp. PCC6803 leads to the activation of the cryptic psbG2 gene.

The genes psbG1 and psbG2 in the cyanobacterium Synechocystis sp. PCC6803 are homologous. The psbG1 gene is located on the chromosome and is part of the ndhC--psbG1--ORF157 operon, while psbG2 is located on a plasmid and is not flanked by equivalent ndhC or ORF157 genes. Mutants in which psbG1 is deleted grow well under autotrophic conditions, while their growth is impeded in mixotrophic medium. These results argue against a functional role for psbG1 in photosynthesis, i.e. photosystem II, and are more compatible with a function in respiration. The psbG2 gene is not transcribed in wild-type cells, but in psbG1 mutants the insertion of DNA sequences in close proximity to the psbG2 reading frame has led to transcriptional activation of psbG2. Thus, psbG2 represents an example of a cryptic gene, similar to those found in other bacteria.

Blotting, Northern↗
PubMed1990

Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin.

We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.

Amino Acid Sequence↗
PubMed1990

Maize chloroplast RNA polymerase: the 180-, 120-, and 38-kilodalton polypeptides are encoded in chloroplast genes.

Prominent polypeptides with apparent molecular masses of 180, 120, 85, and 38 kDa are found in an extensively purified preparation of maize chloroplast DNA-dependent RNA polymerase that retains the capacity to initiate transcription of the cloned chloroplast gene rbcL correctly and the requirement for a supercoiled DNA template for specific and active transcription. Amino-terminal amino acid sequences of the 180-, 120-, and 38-kDa polypeptides have been determined and found to correspond precisely to the sequences deduced from the 5' ends of the maize chloroplast rpoC2, rpoB, and rpoA genes, respectively. These experiments show that these chloroplast rpo genes encode the prominent polypeptides in the highly enriched maize chloroplast RNA polymerase preparation and support the conclusion that these polypeptides are functional components of the enzyme. The rpoB, rpoC1, and rpoC2 genes have been mapped on the maize chloroplast chromosome.

Amino Acid Sequence↗
PubMed1990

Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas.

Transcription from modified chloroplast genes has been studied in vitro, but only with the recently developed ability to stably introduce foreign DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it become possible to do so in vivo. Cloned chloroplast DNA sequences, into which had been inserted chimeric genes composed of the GUS coding sequence reporter under transcriptional control of chloroplast promoters for the C. reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on microprojectiles. These constructs become integrated into chloroplast chromosomes by homologous recombination. RNA gel blot analyses demonstrated that a single major beta-glucuronidase (GUS)-hybridizing transcript accumulates in each chloroplast transformant. We have found that: (1) Transcription of the chimeric gene begins at the same site as in the corresponding endogenous chloroplast gene; (2) the rates of transcription in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to other genes are the same as those for the endogenous atpA and atpB genes, respectively, indicating that these promoters are fully functional despite being fused to a foreign gene and being at an alien location on the chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the expected rate, suggesting that other features are required for optimal activity of this promoter; and (4) 22 base pairs upstream of the 5' end of the atpB:GUS transcript in the atpB promoter element is sufficient to confer wild-type levels of promoter activity.

Animals↗
PubMed1990

Alternative promoters are used for genes within maize chloroplast polycistronic transcription units.

Many chloroplast genes are co-transcribed in polycistronic transcription units that give rise to numerous overlapping RNAs, but the significance of this pattern of transcript accumulation is not understood. An analysis of the transcripts of the adjacent and divergent maize psbE-psbF-psbL-ORF40 and ORF31-petE-ORF42 gene clusters indicates that transcription initiation at alternative promoters contributes to the generation of overlapping RNAs for both clusters. Furthermore, developmentally varying transcript ratios for the ORF31-petE-ORF42 gene cluster are determined at least in part by selective promoter usage. During light-induced plastid maturation, increased levels of primarily monocistronic petE transcripts accumulate from a promoter upstream of the internal petE gene. Dark-predominant and non-light-responsive bi- and tricistronic transcripts result from transcription initiation upstream of ORF31, the proximal gene of the cluster. In addition to the transcriptional overlap within gene clusters, divergent transcription units for the two gene clusters overlap and reciprocal antisense RNAs accumulate. The organization of the transcription units in this region raises the possibility of promoter interdependence or other functional interaction between transcription units.

Amino Acid Sequence↗