scholarly journals Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting

1996 ◽  
Vol 320 (3) ◽  
pp. 769-775 ◽  
Author(s):  
Gisele LOBO-HAJDU ◽  
Hans-Peter BRAUN ◽  
Nancy ROMP ◽  
Leslie A. GRIVELL ◽  
Jan A. BERDEN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Direct Sequencing ◽  
Single Copy ◽  
Cdna Clones ◽  
Mitochondrial Targeting ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Isolated Mitochondria ◽  
Mitochondrial Intermediate Peptidase

cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

scholarly journals Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

1997 ◽  
Vol 325 (2) ◽  
pp. 441-447 ◽  
Author(s):  
Ana CAMACHO ◽  
Rosalía ARREBOLA ◽  
Javier PEÑA-DIAZ ◽  
Luis M. RUIZ-PÉREZ ◽  
Dolores GONZÁLEZ-PACANOWSKA
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Leishmania Major ◽  
Active Form ◽  
Single Copy ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Highly Active

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis

1991 ◽  
Vol 11 (8) ◽  
pp. 4022-4035
Author(s):  
A R Kubelik ◽  
B Turcq ◽  
A M Lambowitz
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Cellular Transport ◽  
Missense Mutations ◽  
Reading Frame ◽  
Trna Synthetases ◽  
Cytosolic Protein

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

scholarly journals Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 125-133 ◽  
Author(s):  
N L Glass ◽  
L Lee
Keyword(s):  
Neurospora Crassa ◽  
Mating Type ◽  
Single Copy ◽  
Open Reading Frame ◽  
Sexual Cycle ◽  
Reading Frame ◽  
Heterokaryon Incompatibility ◽  
Functional Regions ◽  
A Genome ◽  
Ascospore Formation

Abstract In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.

Molecular analysis of a Neurospora crassa gene expressed during conidiation

1988 ◽  
Vol 8 (6) ◽  
pp. 2411-2418
Author(s):  
A N Roberts ◽  
V Berlin ◽  
K M Hager ◽  
C Yanofsky
Keyword(s):  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Group Iv ◽  
Molecular Organization ◽  
Cdna Clones ◽  
Developmental Pathway ◽  
Reading Frame ◽  
Polyadenylation Sites ◽  
Flanking Regions

The asexual developmental pathway in the life cycle of the filamentous fungus Neurospora crassa culminates in the formation of spores called conidia. Several clones of genomic Neurospora DNA have been isolated that correspond to mRNA species expressed during conidiation and not during mycelial growth (V. Berlin and C. Yanofsky, Mol. Cell. Biol. 5:849-855, 1985). In this paper we describe the characterization of one of these clones, named pCon-10a. This clone contains two genes, con-10 and con-13, which are induced coordinately during the later stages of conidiation. The two genes are separated by 1.4 kilobases of DNA; they are located on linkage group IV and are transcribed from the same strand of DNA. The molecular organization and sequence of one of these genes, con-10, and its flanking regions are presented. Full-length cDNA clones for con-10 also were isolated and sequenced, and transcription-initiation and polyadenylation sites were defined. The con-10 gene contains an open reading frame interrupted by two small introns and encodes an 86-amino-acid residue polypeptide that is both hydrophilic and weakly acidic. Expression of the con-10 gene in various mutants defective at different stages of conidiation indicates that it plays a role after aerial hyphal development. Possible functions, organization, and regulation of conidiation-specific genes are discussed.

Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA

1993 ◽  
Vol 105 (2) ◽  
pp. 589-600 ◽  
Author(s):  
T. Maekawa ◽  
R. Kuriyama
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Functional Organization ◽  
Spindle Pole ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Globular Domain ◽  
Calculated Molecular Mass ◽  
Mammalian Cell Lines ◽  
Cell Biol

Using a human autoantibody, SP-H, we identified a 200–230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta lambda gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an alpha-helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central alpha-helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992) J. Cell Biol. 116, 1303–1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992) J. Cell Biol. 116, 1395–1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Jan Cornelissen ◽  
Inky DeBaere ◽  
Marta Steiner-Mosonyi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Messenger Rna ◽  
Coiled Coil ◽  
Cdna Clones ◽  
Exon 2 ◽  
Reading Frame ◽  
Coiled Coil Domain ◽  
Acid Protein ◽  
Apoptosis Protein

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.

scholarly journals cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex

1993 ◽  
Vol 294 (2) ◽  
pp. 589-593 ◽  
Author(s):  
J Cheng ◽  
K J Macon ◽  
J E Volanakis
Keyword(s):  
Major Histocompatibility Complex ◽  
Amino Acid ◽  
Tandem Repeats ◽  
Cdna Clones ◽  
Major Histocompatibility ◽  
Class Iii ◽  
Human Major Histocompatibility Complex ◽  
Calculated Molecular Mass ◽  
Mouse Cdna ◽  
Histocompatibility Complex

The RD gene, initially defined in the mouse, has been mapped between the Bf and C4A genes in the human major histocompatibility complex class III region. Using the mouse cDNA as a probe, we isolated and sequenced human RD cDNA clones. The composite nucleotide sequence consisted of 1301 nucleotides, excluding a poly(A) tail at the 3′ end. It contained a single open reading frame encoding a polypeptide of 380 amino acid residues with a calculated molecular mass of 42274 Da. The most striking structural feature of the deduced amino acid sequence is a region consisting entirely of 24 tandem repeats of an Arg-Asp (or Glu) dipeptide. The human RD cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and used to produce antisera in rabbits. Western blot analysis and immunoprecipitation of lysates of biosynthetically labelled HeLa cells indicated that RD is a 44 kDa nuclear protein.

Glycine cleavage enzyme complex: Rabbit H-protein cDNA sequence analysis and comparison to human, cow, and chicken

2000 ◽  
Vol 78 (6) ◽  
pp. 725-730 ◽  
Author(s):  
Francis Choy ◽  
Lisa Sharp ◽  
Derek A Applegarth
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Matrix Protein ◽  
Protein Gene ◽  
Mitochondrial Targeting ◽  
Reading Frame ◽  
Cleavage Enzyme ◽  
Glycine Cleavage ◽  
H Protein ◽  
Sequence Similarities

The H-protein is one of the four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex, the major degradative pathway of glycine. We have isolated the full-length cDNA of the H-protein gene from the rabbit (Oryctolagus caniculus) by reverse transcription of liver poly-A mRNA and determined its nucleotide sequence (GenBank Acc. No. BankIt 318281 AF 231451). Similar to that in human, the rabbit H-protein gene possesses a 519-bp open reading frame that translates a 173-amino-acid (aa) protein. This reading frame is comprised of a 48-aa mitochondrial targeting sequence and a 125-aa residue that constitutes the mature mitochondrial matrix protein. In the mature protein region, there is a 95.5% nucleotide and 98.4% amino-acid sequence similarity to human. This conservation was also noted in the mature protein of the cow (Bos taurus) and chicken (Gallus domesticus), where there are a 94.1% and 85.3% nucleotide similarities, and 95.2% and 85.6% amino-acid sequence similarities, respectively. However, the targeting region is not as well conserved. Comparison of the rabbit targeting sequence to that in human, cow, and chicken reveals 84.0%, 79.2%, and 72.9% nucleotide, and 72.9%, 75.0%, and 54.2% amino-acid sequence similarities, respectively. These findings suggest that within the H-protein gene, the regions encoding the mitochondrial targeting and matrix protein may have evolved differently. Gene diversification in the former may reflect the species specificity in targeting proteins destined for the mitochondria, whereas homology in the latter suggests a very similar structure-function of the mature H-protein among these species. This homology in structure-function likely accounts for the observation that non-human H-protein can replace the human protein in the activity assay of the glycine cleavage enzyme system. This includes the biochemical diagnosis of non-ketotic hyperglycinemia (NKH) resulting from defects other than the H-protein, e.g., mutation(s) in the P-protein.Key words: glycine cleavage enzyme, H-protein, sequence comparison, non-ketotic hyperglycinemia.

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