A Five-Base-Pair-Deletion in the Gene for the Large Subunit Causes the Lesion in the Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Plastome Mutant Sigma of Oenothera hookeri

The gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) has been mapped on the Oenothera hookeri plastid chromosome. It is located close to the gene for the herbicide-binding “32 kd” protein of the photosystem II reaction center (psbA), at a position different from that found in the ancestral angiosperm type of plastid chromosomes, due to an inversion in the large single-copy region. The gene codes for a polypeptide of 475 amino acid residues corresponding to a molecular mass of 52.7 kd. The deduced amino acid composition diverges by 4.8% from the amino acid sequence of the spinach protein and by 8.2% from that of maize. The corresponding nucleotide sequences differ by 8.5 % and 15 % from each other. The rbcL gene of the RuBPcase/oase-deficient Oenothera plastome mutant sigma contains a TTAAC deletion at amino acid residues 270/271 which introduces a frame shift and an amber stop codon seven triplets later. This lesion which probably arose by slipped mispairing is consistent with the previously observed, virtually full-length mRNA that is decoded into a truncated large subunit polypeptide of approximately 30 kd in vitro and in vivo.     

Chloroplast gene for Mr 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences

The gene for the Mr 32000 herbicide binding polypeptide of photosystem II has previously been mapped to the 5 kbp EcoRI fragment Eco I of Euglena gracilis chloroplast DNA. The nucleotide sequence of 3324 bp of Eco I, containing the psbA locus, has been determined. This locus encodes a polypeptide of 345 amino acids which is co-linear with, and has 86% derived amino acid sequence homology to sequences derived from four higher plants chloroplast psbA loci. The Euglena psbA gene contains four introns of size 435, 443, 434, and 617 bp. The four introns have conserved boundary sequences of the type previously described in the Euglena chloroplast gene (rbcL) for the large subunit of ribulose-1,5-bisphosphate carboxylase (Koller et al., Cell 36, 545-553, 1984).     

Two exons encode the calmodulin-binding domain in the mouse phosphorylase kinase catalytic subunit gene

The catalytic subunit, γ, of phosphorylase kinase contains two calmodulin-binding sequences that define a domain in γ that is homologous to the troponin-C-binding domain in troponin I. The homology is based on both sequence and functional similarities. To account for this homology, it has been proposed that the calmodulin-binding sequences in γ and the troponin-C-binding domain in troponin I have evolved from a common ancestor. We investigated this possibility by comparing the exon structure of the γ gene with that of the troponin-I gene over their homologous domains. In the quail troponin-I gene, it is known that the entire troponin-C-binding domain is encoded by a single exon. However, two exons are found to encode the calmodulin-binding domain in the γ gene from mouse. This result indicates that convergent evolution may be responsible for the sequence and functional similarities between the homologous domains in troponin I and γ.     

Chlorella sorokiniana rbcL基因的克隆与序列分析及其与18S rRNA基因在分类学上的比较

Rubisco大亚基基因(rbcL)广泛用于系统学分析中,在本文中以Chlorella sorokiniana CS-01叶绿体基因组DNA为模板,PCR扩增rbcL全长编码区序列,序列分析表明:该片段全长1428 bp,其中包括1425 bp的编码区序列,编码475个氨基酸,经BLAST比对发现同源性最高的为Chlorella sp.IFRPD 1018,同源性达到99.2%。同时构建系统发育树,结果显示Chlorellasorokiniana CS-01与Chlorella sp.IFRPD 1018在同一分支中。18S rDNA序列分析表明:该片段全长1740 bp,经BLAST比对发现同源性最高的为Chlorella sorokiniana,同源性达到99.7%,构建系统发育树显示Chlorella sorokiniana CS-01与两株Chlorellasorokiniana在同一分支中,支持率达到100%。但是18S rDNA序列构建的系统发育树鉴定的主要分支很少,即使鉴定出分支,该分支的支持率也比较弱,而rbcL基因序列构建的系统发育树则分支清晰且支持率较高。可见18S rDNA序列比rbcL...     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.     

The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.     

Cloning and structural analysis of DNA encoding an A2B1a subunit of glycinin

The partial DNA sequence of a glycinin gene in a genomic clone and a homologous cDNA clone were determined. They have nearly identical nucleotide sequences and encode the basic polypeptide and part of the acidic polypeptide for an A2B1a glycinin subunit. The protein primary structure deduced from the DNA sequence is in close agreement with the amino acid sequence of the subunit determined chemically and confirms assignment of part of the amino acid sequence in the basic component where we were able to establish an overlap using conventional approaches. The coding part of the basic subunit is interrupted by a 625-base pair A + T-rich intron whose boundaries correlate with the established consensus sequences for the exon-intron junctions. Comparison of the nucleotide sequence of the basic subunit of pea legumin gene with that of the gene for A2B1a subunit reveals 70% homology in coding regions, although there is considerably less in the 3'-flanking regions.     

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.     

Different splice site utilization generates diversity between the rat and human pancreatic polypeptide precursors

Pancreatic polypeptide is derived from a polyprotein precursor molecule. Although the amino acid sequences specifying the signal peptide and pancreatic polypeptide are well conserved between the rat and the human, the carboxy-terminal amino acid sequences of the precursors are highly divergent. To better understand the molecular basis of the divergence between the rat and human C-terminal peptides, we have determined the nucleotide sequence of the rat pancreatic polypeptide gene. A comparison between the primary structures of the rat and human genes reveals that the heterogeneity of the C-terminal peptides can be explained in large part by a frameshift mutation and the utilization of an alternative splice donor site in the third exon of the rat gene. As a consequence of the displaced splice site, part of the third exon of the rate gene is homologous to the sequence in the third intron of the human gene. Our results suggest that the rat and human pancreatic polypeptide genes arose from a common ancestral gene, and that differences in the C-terminal domains of the precursor reflect less strict evolutionary constraints than those imposed upon the amino-terminal domains of the precursor.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.