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At least 343 records · Page 19Linked to original sources
PubMed1997

Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase--involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway.

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A full-length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQS1 gene product, SQS1. RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQS1 isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using [3H]farnesyl-P2 and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of polygalacturonase genes (pecA and pecB) from Aspergillus flavus.

Two genes, pecA and pecB, encoding endopolyglacturonases were cloned from a highly aggressive strain of Aspergillus flavus. The pecA gene consisted of 1,228 bp encoding a protein of 363 amino acids with a predicted molecular mass of 37.6 kDa, interrupted by two introns of 58 and 81 bp in length. Accumulation of pecA mRNA in both pectin- or glucose-grown mycelia in the highly aggressive strain matched the activity profile of a pectinase previously identified as P2c. Transformants of a weakly aggressive strain containing a functional copy of the pecA gene produced P2c in vitro, confirming that pecA encodes P2c. The coding region of pecB was determined to be 1,217 bp in length interrupted by two introns of 65 and 54 bp in length. The predicted protein of 366 amino acids had an estimated molecular mass of 38 kDa. Transcripts of this gene accumulated in mycelia grown in medium containing pectin alone, never in mycelia grown in glucose-containing medium, for both highly and weakly aggressive strains. Thus, pecB encodes the activity previously identified as P1 or P3. pecA and pecB share a high degree of sequence identity with polygalacturonase genes from Aspergillus parasiticus and Aspergillus oryzae, further establishing the close relationships between members of the A. flavus group. Conservation of intron positions in these genes also indicates that they share a common ancestor with genes encoding endopolyglacturonases of Aspergillus niger.

Amino Acid Sequence↗
PubMed1985

Replacement variant histone genes contain intervening sequences.

The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cycle.

Age Factors↗
PubMed1986

The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

Actins↗
PubMed1986

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Amino Acid Sequence↗
PubMed1988

A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.

Amino Acid Sequence↗
PubMed2001

Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog.

Mago Nashi, a protein initially shown to be essential in the development of the Drosophila oocyte, is highly conserved among species and shows no homology to any other known cellular proteins. Here we report the nucleotide sequence of a cDNA and a partial gene that encode rice Mago Nashi protein homologs. In addition, we present the tissue-specific expression pattern of mago nashi at the level of RNA and protein. The rice Mago Nashi protein shares at least 73% amino acid identity with all known animal homologs. Genomic DNA gel blot analysis indicates that two copies of the mago nashi gene exist in the rice genome, one of which has identical intron positions to those found in an Arabidopsis homolog. mago nashi is expressed in root, leaf and developing seed tissue as determined by RNA and protein gel blot analysis. Evidence from Drosophila, Caenorhabditis elegans and human studies of Mago Nashi suggests that a major function of this protein is its involvement in RNA localization. The highly conserved amino acid sequence of all Mago Nashi protein homologs across kingdoms suggests that the plant version of this protein may similarly be involved in RNA localization.

Amino Acid Sequence↗
PubMed2004

An unappreciated role for RNA surveillance.

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo. RESULTS: We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA. CONCLUSIONS: For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+--calpain-10, the CDC-like kinases (CLKs) and LARD--show how previous experimental results may be understood in light of NMD.

Alternative Splicing↗
PubMed1999

Characterization of the chalcone synthase genes expressed in flowers of the common and Japanese morning glories.

The CHS genes encoding chalcone synthase for flavonoid biosynthesis in the common and Japanese morning glories comprise a multigene family. Among these Ipomoea CHS genes, the CHS-D gene is the most abundantly expressed in the pigmented young flower buds and is primarily responsible for flower pigmentation. Majority of the remaining CHS transcripts in the flower buds are produced from the CHS-E gene. We characterized the genomic DNA segments of these CHS-D and CHS-E genes. Both genes have two exons with identical intron positions and carry several copies of two mobile element-like sequences with short terminal inverted repeats, MELS3 and MELS6 of around 200-300 bp. Small tandem repeats were also found in these CHS gene regions. The CHS-D and CHS-E genes are expressed predominantly in flower limbs and tubes, respectively. These structural and functional features and their evolutionary implications are discussed.

Acyltransferases↗
PubMed2001

Genomic organization of the murine G protein beta subunit genes and related processed pseudogenes.

The functional significance of heterotrimeric guanine nucleotide binding protein (G protein) for the many physiological processes including the molecular mechanisms of drug addiction have been described. In investigating the changes of mRNA expression after acute psychostimulant administration, we previously identified a cDNA encoding a G protein beta1 subunit (Gbeta1) that was increased up to four-fold in certain brain regions after administration of psychostimulants. The mouse Gbeta1 gene (the mouse genetic symbol, GNB1) was mapped to chromosome 4, but little was known of its genetic features. To characterize the GNB1 gene further, we have cloned and analyzed the genomic structures of the mouse GNBI gene and its homologous sequences. The GNBI gene spans at least 50 kb, and consists of 12 exons and 11 introns. The exon/intron boundaries were determined and found to follow the GT/AG rule. Exons 3-11 encode the Gbeta1 protein, and the exon 2 is an alternative, resulting in putative two splicing variants. Although intron 11 is additional for GNBI compared with GNB2 and GNB3, the intron positions within the protein coding region of GNB1, GNB2 and GNB3 are identical, suggesting that GNB1 should have diverged from the ancestral gene family earlier than the genes for GNB2 and GNB3. We also found the 5'-truncated processed pseudogenes with 71-89% similarities to GNBI mRNA sequence, suggesting that the truncated cDNA copies, which have been reverse-transcribed from a processed mRNA for GNB1, might have been integrated into several new locations in the mouse genome.

Animals↗
PubMed2003

[Gene families of G protein subunits].

G proteins are heterotrimers composed of alpha, beta, and gamma subunits. So far 16 alpha, 6 beta, and 12 gamma subunit genes have been described in mammals. G protein subunit gene families are comparatively conservative and primitive, with all of which exon-intron junctions conform to the GT-AG rule except for one alternative splice site at 5'-end of exon 4 in Gnas which uses GT-TG junction. In the coding regions, exon-intron structures and intron positions among each of the subunit gene families are comparatively conserved. Most of the G protein genes have some characteristics of housekeeping genes. Analysis of distribution of G protein subunit genes in mammalian genomes reveals a tendency of clustering, with five pairs of the alpha subunit genes arranging as tandem duos.

Alternative Splicing↗
PubMed1990

Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the human hepatic lipase gene.

Overlapping bacterial phage and cosmid genomic clones were isolated spanning an area of approximately 60 kilobases that contains the human hepatic lipase (HL) gene. It is composed of 9 exons spanning approximately 35 kilobases of DNA. The entire coding regions, the 5'-flanking sequences, and the exon-intron junctions were sequenced. The intron positions correspond to those of human lipoprotein lipase and canine pancreatic lipase, supporting the concept that these genes constitute a dispersed gene family of lipases and have evolved by duplication of a common ancestral gene. A region of the HL gene, which displays a significant homology with various other lipolytic enzymes and contains the putative catalytic site serine residue of HL, was encoded by exon 4. A major transcription start site of the human HL gene was located by primer extension analysis, 43 nucleotides upstream of the translation initiation codon. Two possible promoter elements were located 25 and 63 nucleotides upstream of the transcription initiation site: a "TATA" box-like sequence, TAATA, and a sequence found in the promoter region of many liver-specific genes, AGGTTAATTATTAAT. In addition, sequences homologous to glucocorticoid and cAMP-responsive elements were identified in the 5'-nontranscribed region.

Amino Acid Sequence↗
PubMed1987

Exon structure of a mannose-binding protein gene reflects its evolutionary relationship to the asialoglycoprotein receptor and nonfibrillar collagens.

Cloned cDNAs encoding mannose-binding proteins isolated from rat liver have been used to isolate one of the genes encoding this group of proteins. This gene, which encodes the minor form of binding protein (designated MBP-A), has been characterized by sequence analysis. The protein-coding portion of the mRNA for the MBP-A is encoded by four exons separated by three introns. The NH2-terminal, collagen-like portion of the protein is encoded by the first two exons. These exons resemble the exons found in the genes for nonfibrillar collagens in that the intron which divides them is inserted between the first two bases of a glycine codon and the exons do not have the 54- or 108-base pair lengths characteristic of fibrillar collagen genes. The carbohydrate-binding portion of MBP-A is encoded by the remaining two exons. This portion of the protein is homologous to the carbohydrate-recognition domain of the hepatic asialoglycoprotein receptor, which is encoded by four exons. It appears that the three COOH-terminal exons of the asialoglycoprotein receptor gene have been fused into a single exon in the MBP-A gene. The organization of the MBP-A gene is very similar to the arrangement of the gene encoding the highly homologous pulmonary surfactant apoprotein, although one of the intron positions is shifted by a single amino acid. The 3' end of a mannose-binding protein pseudogene has also been characterized.

Amino Acid Sequence↗
PubMed1993

Protein anatomy: functional roles of barnase module.

Globular proteins are composed of several modules that are contiguous polypeptide segments of compact conformation. Module boundaries are closely correlated with the intron positions of genes that encode proteins. The modules may thus have a one-to-one correspondence with exons in primordial genes. They may also be vestiges of polypeptide segments that initially appeared as primordial proteins in prebiological evolution. Clarification as to whether modules disconnected from one another have functional potentiality may validate these possibilities. Thus, in this study, each module of a protein was synthesized and assessed for functional potentiality. For this purpose, barnase, a bacterial ribonuclease, was decomposed into six modules (M1-M6), which were examined to determine whether they have an affinity for RNA and RNase activity. M2, M3, and M6, all of which form a shallow but wide cavity for RNA binding in native barnase, were found to bind to RNA and to possess RNase activity. However, M1 and M5, which support the other modules from the back side, and M4 did not bind to RNA and had no RNase activity. Protein modules with catalytic functions are described in this paper for the first time. That some modules of barnase possess catalytic activity indicates that protein modules may possibly have functioned as primitive catalysts in prebiological evolution.

Amino Acid Sequence↗
PubMed1993

A nuclear lamin of the nematode Caenorhabditis elegans with unusual structural features; cDNA cloning and gene organization.

This report describes the characterization of the nuclear lamin CeLam-1 of the nematode Caenorhabditis elegans by molecular analysis of the corresponding complete cDNA and gene sequences. The primary structure of CeLam-1, representing only the third non-vertebrate lamin sequence currently known, follows essentially the features displayed by the B-type lamins of vertebrates and Drosophila. The nematode lamin shows, however, some exceptional properties. First, it lacks the SPTR sequence in front of the coil 1a domain which constitutes the major mitotic cdc2 kinase phosphorylation site. Second, two prominent deletions occur in the CeLam-1 sequence. One eliminates 14 amino acid residues from the coil 2 domain. A larger deletion of approximately 25 residues results in the shortest lamin tail domain documented so far. The latter corresponds to a region which varies considerably in sequence from highly acidic in vertebrate B-type lamins to rather basic in Drosophila lamin Dmo. CeLam-1 is encoded by a single 2.3 kb mRNA which is abundantly expressed in mixed-stage worm populations. The 5'-end of the mRNA is generated by trans-splicing to the SL1 leader sequence. The CeLam-1 gene extending over 2.7 kb is located on chromosome I. The gene is composed of 6 exons and 5 short introns, which all interrupt the coding sequence. Surprisingly, none of the intron positions has a counterpart in either the Drosophila lamin Dmo or the vertebrate lamin genes.

Amino Acid Sequence↗
PubMed1996

Nucleotide structure and characterization of the murine blood coagulation factor VII gene.

The gene encoding murine coagulation factor VII (fVII) has been cloned. Seven introns and eight exons are present, with the introns positioned as splice junctions between the major domain units of the protein. A total of 11,748 bp of the gene was sequenced, and included 1,077 bp of a 5'-flanking region, in which several high probability binding sites for liver transcription factors were present, as well as a CCAAT sequence and possible GC boxes. Primer extension analysis revealed that the major transcription start site was positioned only 9 residues upstream of the ATG initiation codon, thus providing a very short 5'-untranslated region of the gene. The sequence of the CAP site in the murine fVII gene matched exactly the consensus eukaryotic sequence. A total of 1,484 bp of 3'-flanking nucleotides included a probable polyadenylation site (ATTAAA) and an appropriately positioned downstream consensus sequence (AGTGTTTC) for the efficient formation of a 3' terminus of mRNA. These results indicate that all elements are present for liver-based transcription of the gene for murine factor VII. The sequence and restriction endonuclease map of this gene will facilitate construction of fVII deficient mice and mice containing mutant fVII genes.

Amino Acid Sequence↗
PubMed1996

Nucleotide sequence of two actin genes of Lumbricus terrestris.

Two actin genes (act1-LUM and act2-LUM) of the annelid Lumbricus terrestris are characterised. Both genes reveal a single intron of 632 or 696 bp, respectively, located at exactly the same position. This intron position is currently the only intron position shared by some protostomic and most deuterostomic actin genes.

Actins↗