Biomedical subjects
Publications and source records attributed to L Bogorad.
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We have cloned a complementary DNA for pre-apocytochrome c-552 from Chlamydomonas reinhardtii. The deduced sequence of the mature protein shows high homology to those of cytochromes c-553 from cyanobacteria. Its homology to mitochondrial cytochrome c or bacterial photosynthetic cytochrome c2 is lower and appears to be concentrated in sequences around amino acids involved in the interaction with heme. With respect to primary sequence, the "transit sequence" for cytochrome c-552 appears to show no homology to other transit sequences for nuclear encoded chloroplast proteins. However, based on analogy to transit sequences for other proteins (Daldal, F., Cheng, S., Applebaum, J., Davidson, E., and Prince, R. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2012-2016; Goldschmidt-Clermont, M., and Rahire, M. (1986) J. Mol. Biol. 191, 421-432; Smeekens, S., de Groot, M., van Binsbergen, J., and Weisbeek, P. (1986) Cell 46, 365-375) the transit sequence of cytochrome c-552 can be divided into envelope-traversing and thylakoid-traversing domains. Cytochrome c-552 appears to encoded by a single nuclear gene in C. reinhardtii. The gene is expressed exclusively in Cu(II)-deficient cells.
Maize seedlings contain several RNA species complementary to the rpS4 coding strand of the maize chloroplast ribosomal protein gene rpS4. All of these have the same 5' end about 182 bp upstream of the translation start codon for the protein S4. Northern and S1 nuclease analyses of RNA isolated from seedlings at different stages of greening show that the size of the pool of rpS4 transcripts does not change significantly upon illumination of dark-grown seedlings. The rpS4 gene has also been analyzed by in vitro transcription using maize chloroplast RNA polymerase preparations. The site of initiation in vitro has been mapped by S1 nuclease analysis to the same location as the 5' terminus of in vivo transcripts. A sequence resembling other plastid promoters occurs just upstream of this initiation site. The sensitivity of in vitro transcription to DNA template superhelicity has been assessed for the rpS4 gene promoter; its negative superhelicity-transcription rate profile resembles that of rbcL.
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Ac-208, a plastocyanin-deficient strain of Chlamydomonas reinhardtii, lacks the mRNA for pre-apoplastocyanin although Southern analyses indicate no gross deletion in the single gene for pre-apoplastocyanin. The alternative electron carrier, cytochrome c-552, is expressed only in Cu(II)-deficient cells of the mutant. Thus, the mutant strain can grow photoautotrophically in Cu(II)-deficient but not in Cu(II)-sufficient medium. This indicates that the absence of plastocyanin is the only defect (with respect to photosynthesis) in ac-208. Phenotypic revertants of ac-208 are capable of photoautotrophic growth by virtue of constitutive [with respect to Cu(II)] synthesis of cytochrome c-552. The revertant phenotype is attributed to a decreased internal concentration of Cu(II). This indicates that the Cu(II)-dependent expression of cytochrome c-552 synthesis is affected directly by the concentration of Cu(II) rather than indirectly by the level of plastocyanin in the cell.
Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA.
The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.
In order to gain a better understanding of the interaction between the chloroplast and nuclear genomes in controlling the expression of plastid genes and the biosynthesis of chloroplast proteins, maize (Zea mays) nuclear gene mutant hcf(*)-38, in which alpha and beta subunits of coupling factor one (CF(1)) are almost completely missing was studied. The mutant possesses all the other subunits of CF(1) but several peptides of photosystem II are present in reduced amounts. A competitive hybridization experiment showed the presence of the same plastid mRNA species in mutant and wild-type plants except for slightly lower levels of some transcripts in the mutant. Northern hybridization and dot blot hybridization experiments showed the features of transcripts for alpha and beta subunits of CF(1) in the mutant to be similar to those in the wild-type maize although their levels are somewhat lower in the mutant. In vivo and in organello protein labeling experiments with L-[(35)S]Met have shown that alpha and beta subunits of CF(1) are synthesized, assembled into CF(1), and probably associated with thylakoid membranes in mutant plants. It is concluded that they are subsequently degraded.
Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A(+)) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.
When isolated pea plastids are incubated with translation products of poly(A(+)) mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two-dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A(+)) mRNA was isolated from peas at various times during greening and analyzed by this technique. (a) After 48 hours of illumination of dark-grown plants, the relative portion of nuclear encoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course of greening. At least 37 imported polypeptides decreased and 36 increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences in translatable messages were seen between dark- and light-grown plants, they were accompanied by parallel changes in polypeptide abundance.
Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for approximately 90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute approximately 10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts.
Although plastocyanin is not detected in Cu(II)-deficient cells of Chlamydomonas reinhardtii, accumulation of messenger RNA for pre-apoplastocyanin is independent of the concentration of Cu(II) in the medium (Merchant, S., and Bogorad, L. (1986) Mol. Cell. Biol. 6, 462-469). This work shows that the synthesis, transport, and processing of pre-apoplastocyanin also appear to be unaffected in cells grown in Cu(II)-deficient medium. However, the mature protein, presumably formed after import of the precursor into the chloroplast, is rapidly degraded in Cu(II)-deficient cells. The half-life of the mature protein is estimated to be between 16 and 18 min in cells grown in Cu(II)-deficient medium. In cells grown in medium containing Cu(II), the mature protein is stable. The proteolytic activity thus appears to be specific for apoplastocyanin versus plastocyanin and thereby accounts for the absence of accumulated plastocyanin in Cu(II)-deficient cells. This process may be part of a general mechanism designed to remove chloroplast proteins which cannot be utilized.
Exposed portions of the 32 kd chloroplast membrane quinone-binding and triazine herbicide-binding protein of photosystem II have been mapped to the lumenal or to the outer (stromal) surface of the thylakoid by following reactions of antibodies generated against synthetic peptides corresponding to predicted hydrophilic amino acid sequences with normally oriented or everted membrane vesicles. These data have led to the construction of a model with five membrane-spanning domains. The model has been verified, in part, by immunoblots of fragments of the protein produced by trypsin treatment of thylakoids with peptide-specific antibodies. Some of the hydrophilic loops appear to be in close contact with proteins of the oxygen evolving complex of photosystem II inasmuch as their removal increases the antibody reaction.
We recently described a pair of partially homologous maize chloroplast genes, one of which was shown to code for an apoprotein of the P700 chlorophyll a complex of photosystem I (Fish, L.E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). Two chlorophyll-free apoprotein bands from maize chlorophyll-protein complex I (CPI) can be resolved on lithium dodecyl sulfate (LDS)-urea polyacrylamide gels. Proteins in both bands react with antibodies prepared against CPI, but antibodies prepared against two synthetic peptides corresponding to predicted sequences of PSI-A1 react only with the upper band. The presence of products of the two genes, ps1A1 and ps1A2, in CPI was verified by analysis of cyanogen bromide (CNBr) fragments of the lower apoprotein band obtained from LDS-urea polyacrylamide gels by reverse-phase high pressure liquid chromatography. Amino-terminal sequencing of five CNBr fragments indicates that the lower band contains a product of the ps1A2 gene. The possibility of extensive processing was investigated because the apparent molecular masses of the maize CPI proteins are about 58-70 kDa on LDS-polyacrylamide gels rather than the predicted sizes of about 83 kDa. Antibodies against a synthetic peptide corresponding to a predicted sequence in PSI-A1 were used to determine that the amino-terminal end of PSI-A1 is intact beyond about position 52. The amino-terminal CNBr fragment of PSI-A2 was identified by sequencing, indicating that the amino-terminal end of PSI-A2 is not processed. The carboxyl-terminal CNBr fragment of PSI-A2 was also identified by sequencing. These results indicate that the PSI-A1 and PSI-A2 polypeptides are not extensively processed, although some processing at the carboxyl-terminal end has not been ruled out.
An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.
Bundle sheath chloroplasts of maize leaves contain about one-fourth as much light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP-II) as do mesophyll chloroplasts. We have determined that this difference is, in part, the result of differential expression of different LHCP-II genes. We have prepared and partially characterized cDNA clones specific for six LHCP-II genes of maize. Transcripts of these six LHCP-II genes are present at vastly different levels and account for about 95% of total LHCP-II mRNAs in bundle sheath and mesophyll cells of illuminated dark-grown maize leaves. Three genes are preferentially expressed in mesophyll cells, and their mRNAs constitute about 54% of the total LHCP-II transcripts in greening (24 hr) maize leaves. Two genes are expressed equally in bundle sheath and mesophyll cells. Most interestingly, the RNA of one gene that contributes about 8% of the total LHCP-II transcripts in leaves greening for 24 hr is present at a much higher level in bundle sheath than in mesophyll cells. Moreover, immunoblot analysis of maize thylakoids reveals at least five sizes of LHCP-II; these also differ from one another in their relative abundance in bundle sheath and mesophyll cells of developing maize leaves.
Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 X 10(8) cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmid is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV-inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 X 10(-4) and 1.5 X 10(-5), respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.
Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA.