Nucleotide sequence of the gene for pre-apocytochrome f in the spinach plastid chromosome

Summary We have characterized a cloned fragment of the spinach plastid chromosome encoding the gene for apocytochrome f. Northern blot analysis and hybrid selection translation discloses that the gene is expressed. From the nucleotide sequence, we deduce that the protein contains 285 amino acids and an amino-terminal signal sequence of 35 amino acid residues. The calculated molecular mass of pre-apocytochrome f is 35.3 kd. The clustering of hydrophobic residues indicates that the processed protein (31.3 kd) possesses only a single anchoring transmembrane domain close to the C terminus, and that 75% of the polypeptide chain including the heme-binding site protrudes into the thylakoid lumen. This topology resembles that reported for beef heart mitochondrial cytochrome c₁.     

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

Summary A 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.     

Hypothesis for the evolutionary origin of the chloroplast ribosomal protein L21 of spinach

Summary A full size cDNA clone encoding the chloroplast ribosomal protein L21 from spinach is presented. The identity of the clone and the location of the transit peptide processing site were determined by comparison with the N-terminal amino acid sequence of the spinach chloroplast protein CS-L7 previously identified. L21 r-protein sequences from spinach, Marchantia polymorpha and Escherichia coli are compared. Quite surprisingly, the data do not suggest that the rpl21 nuclear gene from spinach was derived through intracellular gene transfer from the chloroplast genome. The possibility of a mitochondrial origin for rpl21 gene of spinach is discussed.     

Pea chloroplast genes encoding a 4kDa polypeptide of photosystem I and a putative enzyme of C1 metabolism

Summary The nucleotide sequence of 3.2 kbp of pea chloroplast DNA located upstream from the petA gene for cytochrome f, and previously reported to contain the gene for a photosystem I polypeptide, has been determined. Three open reading frames of 587, 40 and 157 codons have been identified. Orf40 encodes a highly conserved, hydrophobic, membrane-spanning polypeptide, and is identified as the gene psaI for the 4 kDa subunit of photosystem I. Orf587 is an extended version of the gene zfpA previously identified as encoding a conserved putative zinc-finger protein. The product of orf587 shows extensive homology to an unidentified open reading frame cotranscribed with a gene for folate metabolism in Escherichia coli and local homology to a region of the subunit of rat mitochondrial propionyl-CoA carboxylase. It is suggested that the product of orf587 is an enzyme of C₁ metabolism and is unlikely to be a regulatory DNA-binding protein. Orf157 potentially encodes an unidentified basic protein, but the protein sequence is not conserved in other plants.     

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae

Summary Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T₂ gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T₂ revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T₂ activity than wild-type strains.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.     

Organization and nucleotide sequence of the broad bean chloroplast genes trnL-UAG,ndhF and two unidentified open reading frames

Summary We have determined the nucleotide sequence of a 6.9 kbp BamHI-Xbal fragment of broad bean chloroplasts. Part of this fragment (subfragment BglII-CIal) is known to contain three tRNA genes (trnL-CAA, trnlrUAA and trnF). We have now further identified a gene coding for the third tRNA^Leu isoacceptor (trnL-UAG) which is located close to trnF. The BamHI-Xbal fragment also contains the gene for subunit 5 of NADH dehydrogenase (ndhF) and two unidentified open reading frames (ORFx and ORF48). ORFx shares a high sequence homology with the long reading frames of tobacco (ORF1708), spinach (ORF2131), and liverwort (ORF2136), while ORF48 shares sequence homology with ORF69 of liverwort and ORF55 of tobacco.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

Antidiphtheritic activity of a fragment of the epidermal growth factor precursor

Expression of a DNA fragment coding for the 76–208 domain of protein molecule of the epidermal growth factor precursor, which contains a sequence of 133 N-terminal amino acid residues, was attained by gene engineering and molecular biology methods. The fusion protein was produced with very low efficacy inE. coli cells. It was purified by metal-affinity chromatography and its specific antitoxic activity evaluated in guinea pigs was at least 10⁶ IU/mg protein. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 92–94, January, 1999     

Plastocyanin is encoded by an uninterrupted nuclear gene in spinach

Summary Plastocyanin is a member of photosynthetic electron transport chains that transfers electrons from cytochrome f to the oxidized P₇₀₀ chlorophyll a pigment of the photosystem I reaction center. We have isolated and characterized cDNA- and genomic clones from spinach (Spinacia oleracea) encoding the complete plastocyanin-precursor polypeptide. The amino acid sequence derived from the nucleotide sequence shows that the precursor consists of 168 amino acid residues including a transit sequence of 69 residues. The precursor polypeptide has a predicted M_r of 16,917, the mature protein of 10,413. The available data indicate that plastocyanin derives probably from a single-copy gene. The coding region contains no intron. The size of the mRNA as determined by S1 nuclease protection experiments is approximately 660 nucleotides, although analysis of different cDNA clones suggests that longer RNA species do exist, approaching the size of the mRNA (850 bases) estimated by Northern blot techniques.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Repetitive sequence-mediated rearragements in Chlorella ellipsoidea chloroplast DNA: completion of nucleotide sequence of the large inverted repeat

Summary A 3454 base pair (bp) sequence of the large inverted repeat (IR) of chloroplast DNA (cpDNA) from the unicellular green alga Chlorella ellipsoidea has been determined. The sequence includes: (1) the boundaries between the IR and the large single copy (LSC) and the small single copy (SSC) regions, (2) the gene for psbA and (3) an approximately 1.0 kbp region between psbA and the rRNA genes which contains a variety of short dispersed repeats. The total size of the Chlorella IR was determined to be 15243 bp. The junction between the IR and the small single copy region is located close to the putative promoter of the rRNA operon (906 bp upstream of the-35 sequence on each IR). The junction between the IR and the large single copy region is also just upstream of the putative psbA promoter, 218 bp upstream from the ATG initiation codon. A few sets of unique sequences were found repeatedly around both junctions. Some of the sequences flanking the IR-LSC junction suggest a unidirectional and serial expansion of the IR within the genome. The psbA gene is located close to the LSC-side junction and codes for a protein of 352 amino acid residues. A highly conserved C-terminal Gly is absent. Unlike the psbA of Chlamydomonas species, which contains 2–4 large introns, the gene of Chlorella has no introns. The overall gene organization of the Chlorella IR is very different from that of higher plants, but a similar gene cluster of rrn-psbA is also found in the IR of Chlamydomonas species and in a single copy region of some chlorophyll a/c-containing algae, indicating a common evolutionary lineage of these cpDNAs. The origin and evolution of the IR structure are discussed in the light of these observations.     

The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe

Summary The pho4 gene of the fission yeast Schizosaccharomyces pombe is regulated by thiamin. The nucleotide sequence of this gene is given here and it is shown that it matches the amino acid sequence of thiamin-repressible acid phosphatase, corroborating genetic evidence that pho4 represents the structural gene of this enzyme. The gene codes for a protein of 463 amino acids in length and shows regions of strong similarity with the phosphate-repressible acid phosphatase of Schizosaccharomyces pombe. The enzyme has a cleavable signal sequence 18 amino acids long and carries nine potential N-glycosylation sites.     

Identification of major antigenic proteins ofPasteurella piscicida

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicidarabbit serum from a genomic DNA library ofP. piscicidastrain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins inEscherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins ofP. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein ofP. piscicidawith previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci inE. colifor thedegQanddegSserine protease genes. A sequence in the 3′ non-coding region ofVibrio hollisaethermostable hemolysin gene that is highly homologous with a similar located sequence in thePseudomonas putidap-cresol methylhydroxylase gene is also found in the 3′ non-coding region of thedegShomolog gene of the PPA2.     

rps10, unreported for plastid DNAs,is located on the cyanelle genome of Cyanophora paradoxa and is contranscribed with the str operon genes

Summary rps10, encoding the plastid ribosomal protein S10, is a nuclear gene in higher plants and green algae, and is missing from the large ribosomal protein gene cluster of chlorophyll b-type plastids that contains components of the prokaryotic S10, spc and alpha operons. The cyanelle genome of Cyanophora paradoxa is shown to harbor rps10 as another specific feature of its organization. However, this novel plastid gene is not contiguous with the genes of the S10 operon, but is adjacent to, and cotranscribed with, the str operon, a trait also found in archaebacteria.     

Nucleotide sequences of cDNAs encoding four complete nuclear-encoded plastid ribosomal proteins

Summary The nucleotide sequences of four pea nuclearencoded plastid ribosomal protein cDNAs have been determined. These cDNAs were shown to encode the complete precursor proteins. The transit sequences of the encoded proteins are similar to the transit sequences of other imported proteins being rich in serine and/or threonine and lacking aspartic and glutamic acid. The transit sequences do not, however, have any apparent amino acid sequence similarity with one another or with the transit sequences of other imported proteins. The derived amino acid sequences of the plastid ribosomal proteins were compared to the amino acid sequences of other ribosomal proteins. Significant amino acid sequence similarity was found between Escherichia coli ribosomal proteins L9 and L24 and two of the nuclearencoded pea plastid ribosomal proteins.     

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

Summary Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.The sequence reported in this paper has been submitted to the EMBL/GenBank through DDBJ (accession no, X52525).     

The Complete CDS of the Prion Protein (<Emphasis Type="Italic">PRNP</Emphasis>) Gene of African Lion (<Emphasis Type="Italic">Panthera leo</Emphasis>)

We provide the complete PRNP CDS sequence for the African lion, which is different from the previously published sequence and more similar to other carnivore sequences. The newly obtained prion protein sequence differs from the domestic cat sequence at three amino acid positions and contains only four octapeptide repeats. We recommend that this sequence be used as the reference sequence for future studies of the PRNP gene for this species.     

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA^His (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA^Leu (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.     

Sequencing and Characterisation of p74 Gene in Two Isolates of Anticarsia Gemmatalis MNPV

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.