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PubMed1988

Complementary DNA clones of chicken proto-oncogene c-ets: sequence divergence from the viral oncogene v-ets.

The avian acute leukemia virus E26 induces erythroblastosis and myeloblastosis in chickens. The oncogene of this virus includes sequences derived from the cellular gene designated c-ets, which is normally expressed in lymphoid cells and whose product is a protein of apparent molecular weight ca. 54,000 daltons. Complementary DNA clones representing the major transcript of the chicken c-ets proto-oncogene were isolated from a spleen cell library. Sequence analysis of the cDNA revealed that it contains an open reading frame encoding a polypeptide of 441 amino acids with a molecular weight of 49,932 daltons. This open reading frame can be transcribed and translated in vitro into a 50 kd protein that is specifically immunoprecipitated with antiserum to the v-ets oncogene product. Within the central region of homology between c-ets and v-ets, there are only 5 nucleotide substitutions resulting in 4 amino acid changes. However, coding sequences at the 5' and 3' ends of the v-ets oncogene and the chicken c-ets cDNA differ from one another. These changes may be responsible for the differential functions of c-ets and v-ets in cells of different hematopoietic lineages and may account for the pathogenic properties of the v-ets oncogene.

Amino Acid Sequence↗

PubMed1994

Identification of human melanoma cellular and secreted ligands for galectin-3.

The endogenous human tumor-associated galectin-3 (hL-31) is a functional molecule which acts as a receptor for ligands containing poly-N-acetyllactosamine sequences. However, little is known about its native ligand(s). In order to identify the ligand(s), the human melanoma cell line A375 was metabolically labeled with [3H]glucosamine, and total cell extracts and serum-free conditioned medium of the labeled cells were affinity-purified on immobilized recombinant hL-31 followed by elution with lactose, the specific sugar inhibitor of the lectin. Cellular ligands for hL-31 were found to be composed of the two lysosome-associated membrane proteins, LAMP-1 and LAMP-2, while secreted ligands consisted of two glycoproteins of 98 and 70 kDa. N-terminal protein microsequencing revealed that the 98 kDa and 70 kDa species share the same N-terminal sequence. The functional relevance of these secreted ligands was demonstrated by their ability to inhibit lectin-mediated hemagglutination in a manner similar to the specific sugar inhibitor lactose. Computer-assisted sequence library searches have identified the 98 kDa human melanoma secreted ligand to be the Mac-2-binding protein (Mac-2-BP), also known as the human lung tumor L3 antigen.

Amino Acid Sequence↗

PubMed1987

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide.

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.

Amino Acid Sequence↗

PubMed1993

Coproporphyrinogen oxidase. Purification, molecular cloning, and induction of mRNA during erythroid differentiation.

Coproporphyrinogen oxidase (EC 1.3.3.3), the enzyme involved in the sixth step of heme biosynthesis, was purified to apparent homogeneity from bovine liver; it has a molecular mass of 37,000 daltons. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequences of trypsin-digested peptides were used in a polymerase chain reaction to amplify a 198-base pair fragment of coproporphyrinogen oxidase DNA, using bovine kidney cells cDNA as a starting template. This fragment was used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library. Sequence analysis revealed that coproporphyrinogen oxidase comprises 354 amino acid residues (M(r) 40,647), with a putative leader sequence of 31 amino acid residues, the result being a mature protein of 323 amino acid residues (M(r) 37,255). RNA blot analysis revealed a 3.0-kilobase coproporphyrinogen oxidase mRNA in mouse liver and in MEL cells. Treatment of MEL cells with dimethyl sulfoxide led to an increase in coproporphyrinogen oxidase mRNA within 10 h, the induction reached a maximum at 24 h, and was in parallel with the induction of ferrochelatase mRNA. The cDNA allows for the expression of active coproporphyrinogen oxidase, the activity of which is mainly present in mitochondria of transfected cultured cells, thereby indicating that mammalian coproporphyrinogen oxidase is mitochondrial enzyme.

Amino Acid Sequence↗

PubMed2001

Polymorphism of flagellin A gene in Helicobacter pylori.

AIM: To study the polymorphism of flagellin A genotype and its significance in Helicobacter pylori (H. pylori). METHODS: As the template, genome DNA was purified from six clinical isolates of H. pylori from outpatients, and the corresponding flagellin A fragments were amplified by polymerase chain reaction. All these products were sequenced. These sequences were compared with each other, and analyzed by software of FASTA program. RESULTS: Specific PCR products were amplified from all of these H. pylori isolates and no length divergence was found among them. Compared with each other, the highest ungapped identity is 99.10%, while the lowest is 94.65%. Using FASTA program, the alignments between query and library sequences derived from different H. pylori strains were higher than 90%. CONCLUSION: The nucleotide sequence of flagellin A in H. pylori is highly conservative with incident divergence. This information may be useful for gene diagnosis and further study on flagellar antigen phenotype.

Base Sequence↗

PubMed2000

Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.

Animals↗

PubMed2002

How can we deliver the large plant genomes? Strategies and perspectives.

The first sequenced plant genome, from the small mustard plant Arabidopsis thaliana, was published at the end of 2000. The sequencing of the rice genome is well under way. The sizes of plant genomes vary by a factor of up to 1000, and many important crop plants have genomes that are several times larger than the human genome. To gain insight into the gene toolbox of plant species, numerous large-scale EST sequencing projects have been launched successfully, and analysis procedures are constantly being refined to add maximum value to the sequence data. In addition, an alternative approach to exclude repetitive noncoding DNA and to enrich sequence libraries for gene-containing genomic regions has been developed. This strategy has the potential to deliver information about both genes and regulatory regions outside the transcribed regions.

Arabidopsis↗

PubMed1996

The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays.

The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Base Sequence↗

PubMed2001

Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure.

Of the sequence comparison methods, profile-based methods perform with greater selectively than those that use pairwise comparisons. Of the profile methods, hidden Markov models (HMMs) are apparently the best. The first part of this paper describes calculations that (i) improve the performance of HMMs and (ii) determine a good procedure for creating HMMs for sequences of proteins of known structure. For a family of related proteins, more homologues are detected using multiple models built from diverse single seed sequences than from one model built from a good alignment of those sequences. A new procedure is described for detecting and correcting those errors that arise at the model-building stage of the procedure. These two improvements greatly increase selectivity and coverage. The second part of the paper describes the construction of a library of HMMs, called SUPERFAMILY, that represent essentially all proteins of known structure. The sequences of the domains in proteins of known structure, that have identities less than 95 %, are used as seeds to build the models. Using the current data, this gives a library with 4894 models. The third part of the paper describes the use of the SUPERFAMILY model library to annotate the sequences of over 50 genomes. The models match twice as many target sequences as are matched by pairwise sequence comparison methods. For each genome, close to half of the sequences are matched in all or in part and, overall, the matches cover 35 % of eukaryotic genomes and 45 % of bacterial genomes. On average roughly 15% of genome sequences are labelled as being hypothetical yet homologous to proteins of known structure. The annotations derived from these matches are available from a public web server at: http://stash.mrc-lmb.cam.ac.uk/SUPERFAMILY. This server also enables users to match their own sequences against the SUPERFAMILY model library.

Amino Acid Sequence↗

PubMed2000

Structure, chromosomal localization, and promoter analysis of the human elastin microfibril interfase located proteIN (EMILIN) gene.

Elastin microfibril interfase-located protein (EMILIN) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as the blood vessels, skin, heart, and lung. It occurs with elastic fibers at the interface between amorphous elastin and microfibrils. In vitro experiments suggested a role for EMILIN in the process of elastin deposition. This multimodular protein consists of 995 amino acids; the domain organization includes a C1q-like globular domain at the C terminus, a short collagenous stalk, a region containing two leucine zippers, and at least four heptad repeats with a high potential for forming coiled-coil alpha-helices and, at the N terminus, a cysteine-rich sequence characterized by a partial epidermal growth factor-like motif and homologous to a region of multimerin. Here we report the complete characterization of the human and murine EMILIN gene, their chromosomal assignment, and preliminary functional data of the human promoter. A cDNA probe corresponding to the C terminus of EMILIN was used to isolate two genomic clones from a human BAC library. Sequencing of several derived subclones allowed the characterization of the whole gene that was found to be about 8 kilobases in size and to contain 8 exons and 7 introns. The internal exons range in size from 17 base pairs to 1929 base pairs. All internal intron/exon junctions are defined by canonical splice donor and acceptor sites, and the different domains potentially involved in the formation of a coiled-coil structure are clustered in the largest exon. The 3'-end of the EMILIN gene overlaps with the 5'-end of the promoter region of the ketohexokinase gene, whose chromosomal position is between markers D2S305 and D2S165 on chromosome 2. A 1600-base pair-long sequence upstream of the translation starting point was evaluated for its promoter activity; five deletion constructs were assayed after transfection in primary chicken fibroblasts and in a human rhabdomyosarcoma cell line. This analysis indicates the existence of two contiguous regions able to modulate luciferase expression in both cell types used, one with a strong activatory function, ranging from positions -204 to -503, and the other, ranging from positions -504 to -683, with a strong inhibitory function.

5' Untranslated Regions↗

PubMed1997

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes.

BACKGROUND/AIMS: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exo-erythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study, we have unravelled the nature of the 55 kD protein. METHODS: For the identification of this protein, a 53-58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53-58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7-5.8. RESULTS: Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an antiserum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane. CONCLUSIONS: Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

Amino Acid Sequence↗

PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗

PubMed1999

Isolation and characterization of marsupial IL5 genes.

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.

Amino Acid Sequence↗

PubMed1997

Isolation and characterization of CYP6B4, a furanocoumarin-inducible cytochrome P450 from a polyphagous caterpillar (Lepidoptera:papilionidae).

Papilio glaucus (tiger swallowtail) is a generalist that rarely encounters plants containing furanocoumarins yet is constitutively capable of metabolizing low levels of these highly toxic allelochemicals. In larvae of this species, metabolism of linear (xanthotoxin, bergapten), and angular (angelicin, sphondin), furanocoumarins can be induced up to 30-fold by the presence of xanthotoxin in their diet. Degenerate primers corresponding to conserved amino acid sequences in three insect P450s, Musca domestica (CYP6A1), Drosophila melanogaster (CYP6A2) and Papilio polyxenes (CYP6B1), were used to clone xanthotoxin-induced P450 transcripts from P. glaucus larvae by a reverse transcription-polymerase chain reaction (RT-PCR) strategy. Positive clones encoding the highly conserved F--G-R-C-G P450 signature motif were used to isolate a full-length CYP6B4v1 cDNA from a P. glaucus xanthotoxin-induced cDNA library. Sequence comparisons indicate the P. glaucus CYP6B4v1 protein sequence is 63% and 61% identical, respectively, to the P. polyxenes furanocoumarin-inducible CYP6B1v1 and CYP6B3v1 proteins. Northern analysis indicates that CYP6B4 and related transcripts are highly induced in response to xanthotoxin. Baculovirus-mediated expression of the CYP6B4v1 protein in lepidopteran cell lines demonstrates that this P450 isozyme metabolizes isopimpinellin, imperatorin, and bergapten at high rates, xanthotoxin and psoralen at intermediate rates and angelicin, sphondin, and trioxsalen only at very low rates.

5-Methoxypsoralen↗

PubMed2001

Molecular cloning and characterization of WdPKS1, a gene involved in dihydroxynaphthalene melanin biosynthesis and virulence in Wangiella (Exophiala) dermatitidis.

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Delta disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a beta-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Delta strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Delta strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

Amino Acid Sequence↗

PubMed2000

Human nuclear respiratory factor 2 alpha subunit cDNA: isolation, subcloning, sequencing, and in situ hybridization of transcripts in normal and monocularly deprived macaque visual system.

Nuclear respiratory factor 2 (NRF-2) has been shown to contribute to the transcriptional regulation of a number of subunits of respiratory chain enzymes, including cytochrome c oxidase (CO). Our recent study demonstrated a parallel distribution of the alpha subunit proteins of NRF-2 (NRF-2 alpha) with CO in the monkey striate cortex, and that it can be regulated by neuronal activity. To determine whether this regulation is at the transcriptional level, the present study examined the expression of NRF-2 alpha mRNA in normal and monocularly deprived adult monkeys. A partial NRF-2 alpha cDNA was isolated from a human brain cDNA library. Sequence analysis revealed that it shared 99% identity with the published sequence from human HeLa cells. Riboprobes of NRF-2 alpha was generated and labeled with digoxigenin-11-UTP for in situ hybridization. The expression pattern of NRF-2 alpha mRNA in the normal striate cortex paralleled that of CO activity. It was highly expressed in layers IVC and VI, which contained high levels of CO, and more densely expressed in puffs of layers II and III than in interpuffs. In monkeys monocularly treated with tetrodotoxin for 1 day to 2 weeks, both NRF-2 alpha expression and CO activity were reduced in deprived ocular dominance columns of the visual cortex and in deprived layers of the lateral geniculate nucleus. These data indicate that, in the normal and visually deprived adult monkeys, NRF-2 alpha is regulated by neuronal activity at the transcriptional level.

Amino Acid Sequence↗

PubMed1998

Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants.

The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (ERG6) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in Saccharomyces cerevisiae. The Candida albicans ERG6 gene was isolated by complementation of a S. cerevisiae erg6 mutant by using a C. albicans genomic library. Sequencing of the Candida ERG6 gene revealed high homology with the Saccharomyces version of ERG6. The first copy of the Candida ERG6 gene was disrupted by transforming with the URA3 blaster system, and the second copy was disrupted by both URA3 blaster transformation and mitotic recombination. The resulting erg6 strains were shown to be hypersusceptible to a number of sterol synthesis and metabolic inhibitors, including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin, and brefeldin A. No increase in susceptibility to azoles was noted. Inhibitors of the ERG6 gene product would make the cell increasingly susceptible to antifungal agents as well as to new agents which normally would be excluded and would allow for clinical treatment at lower dosages. In addition, the availability of ERG6 would allow for its use as a screen for new antifungals targeted specifically to the sterol methyltransferase.

Amino Acid Sequence↗

PubMed1985

Computer generation and statistical analysis of a data bank of protein sequences translated from GenBank.

We describe PGtrans, a new and freely available protein sequence databank (2625 sequences, 554198 amino-acids). This data bank is routinely produced by automatic computer translation of the nucleotide sequence library GenBank. The information needed for the translation process (transcriptional orientation, location of coding regions, splice sites and pertinent genetic code) is gathered by the translation program through an "intelligent" scanning of the documentary field of each GenBank entry. Inconsistencies resulting in unexpected termination codons are detected and reported thus allowing the correction of data bank errors. PGtrans is intended as a tool for protein similarity searches. Its reasonable overall size (2 Moctets) makes it suitable for micro-computer environments. Up to date amino-acid composition data and relative abundances of di-, tri-, and tetra-peptides in proteins of known sequences are presented and discussed.

Amino Acid Sequence↗