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PubMed1979

The regulatory region of MS2 phage RNA replicase cistron. IV. Functional activity of specific MS2 RNA fragments in formation of the 70 S initiation complex of protein biosynthesis.

The initiation region of the MS2 replicase cistron can be isolated as a fragment 59 bases in length protected from RNAase by the binding of the coat protein which serves as a translational repressor. This fragment MS2 R(-53 leads to 6) starts 53 bases before the initiation codon and retains full activity in binding ribosomes. We have investigated the functional activity in initiation of a series of fragments from this region variously shortened from the 5'-end. Ribosome protected fragments starting 17 or 21 bases before the AUG are unable to rebind to ribosomes. The shortest fragment which has this activity was produced by partial S1 nuclease digestion and starts 33 to 35 bases before the AUG. The initiation signal comprises some nucleotides between 21 and 33 bases before the initiation codon and the regulatory region responsible for initiation is longer than that protected by the ribosome in the final initiation complex.

Base Sequence↗

PubMed1996

The glycosylated gag protein of MuLV is a determinant of neuroinvasiveness: analysis of second site revertants of a mutant MuLV lacking expression of this protein.

Neuroinvasiveness is a property of all neurovirulent murine retroviruses, although the factors which facilitate infection of the CNS are not understood. We previously showed that mutant MuLV which lack expression of an accessory protein, glycosylated gag, had lost neurovirulence, indicating that this protein may be involved in promoting CNS infection. The mutations were located in the "Kozak" consensus sequence of the initiation codon for this protein. Here it is shown that shortly after inoculation of mice with one of these mutant viruses, revertants emerged which had regained expression of glycosylated gag and had also regained the neuroinvasiveness and neurovirulence exhibited by the wild-type virus. The phenotypic revertants retained the mutations in the "Kozak" consensus sequence but exhibited a G-->A mutation 12 codons downstream from the mutated start site, creating a new initiation codon and a glycosylated gag protein, which was truncated at its N-terminus. Using antibodies specific for glycosylated gag it is shown that the frequency of splenic infectious centers expressing revertant virus increased progressively during the 2 months following inoculation of mutant virus until > or = 50% of the virus-producing cells in the spleen expressed revertant virus. In contrast, although phenotypic revertants were detectable at low frequency after cell-free passage in vitro in M. dunni fibroblasts, there was no evidence for selection. These results indicate that glycosylated gag facilitates virus spread within the spleen and to extra-splenic sites, such as the CNS, and suggest that the protein may function through its interaction with the host.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and nucleotide sequence of the colonization factor antigen I gene of Escherichia coli.

The colonization factor antigen I (CFA/I) gene has been isolated and sequenced. The amino acid sequence of CFA/I deduced from the nucleotide sequence is composed of 170 amino acids. The first 23 amino acids are considered to be the signal peptide of the CFA/I protein since they are not present in the protein sequence. Among the remaining amino acids, only two are different from the protein sequence: amino acid position 76 is an aspartic acid instead of an asparagine, and position 97 is a serine instead of an alanine. The CFA/I gene has a typical Shine-Dalgarno sequence located 10 base pairs (bp) upstream from the initiation codon. The sequence TACAAT located 48 bp upstream from the initiation codon was tentatively designated the -10 sequence of the CFA/I gene promoter. No sequences homologous to the consensus -35 promoter sequence was found. A pair of inverted repeat sequences followed by a stretch of eight A's are located 45 bp downstream from the termination codon of the CFA/I gene; this region may be a rho-independent transcriptional terminator.

Amino Acid Sequence↗

PubMed1992

Characterization of activated and normal mouse Mos gene in murine 3T3 cells.

We have characterized the mouse Mos proto-oncogene product, pp39Mos, in murine fibroblasts. When expressed in NIH3T3 cells under the influence of the long terminal repeat regulatory element from Moloney murine sarcoma virus [NIH(pTS-1) cells], the Mos protein was present in low levels and had a half-life of about 30 min. In extracts from NIH(pTS-1) cells, we detected additional forms of Mos protein that apparently arose from internal initiation codons (p24Mos and p29Mos) or from upstream non-AUG initiation codons (p42Mos and p44Mos). The Mos protein was found to exist in these cells as a phosphoprotein, pp39Mos, and, when immunoprecipitated with an antiserum specific for the Mos N-terminus [anti-Mos(6-24)], had autophosphorylating kinase activity. We found that anti-Mos(6-24) also detected non-Mos protein kinase activity and non-Mos phosphoproteins in addition to p39Mos. We present evidence, on both the RNA and protein levels, that non-transformed mouse 3T3 cells do not express endogenous Mos.

3T3 Cells↗

PubMed1987

In vitro synthesis of vaccinia virus late mRNA containing a 5' poly(A) leader sequence.

We have shown that an extract made from HeLa cells harvested 6 hr after infection with vaccinia virus can transcribe a duplex DNA template containing a late viral gene. S1 nuclease analyses using genomic and synthetic probes indicated that the 5' ends of RNA synthesized in vitro are similar to those of RNA made in vivo and contain 5' poly(A) sequences contiguous with the translation initiation codon. Kinetic analysis of RNA synthesized in vitro demonstrated that a correctly initiated and 5' polyadenylylated product appeared within 5 min after transcription reactions were started. A cis-splicing mechanism of poly(A) addition can be ruled out because the DNA template used in vitro had no poly(dT) sequence and could contain as few as 37 base pairs upstream of the start of the RNA. In addition, we found that a point mutation in the first of two consecutively encoded adenylate residues preceding the ATG initiation codon abolished transcription in vitro. These data are consistent with at least three models: (i) RNA polymerase initiates RNA synthesis with a run of adenylate residues; (ii) a poly(A) primer is used for initiation; or (iii) the poly(A) leader is rapidly and efficiently attached to the RNA by ligation.

Cell-Free System↗

PubMed1985

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Cell Line↗

PubMed2004

[Analysis, identification and correction of some errors of model refseqs appeared in NCBI Human Gene Database by in silico cloning and experimental verification of novel human genes].

We found that human genome coding regions annotated by computers have different kinds of many errors in public domain through homologous BLAST of our cloned genes in non-redundant (nr) database, including insertions, deletions or mutations of one base pair or a segment in sequences at the cDNA level, or different permutation and combination of these errors. Basically, we use the three means for validating and identifying some errors of the model genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS: (I) Evaluating the support degree of human EST clustering and draft human genome BLAST. (2) Preparation of chromosomal mapping of our verified genes and analysis of genomic organization of the genes. All of the exon/intron boundaries should be consistent with the GT/AG rule, and consensuses surrounding the splice boundaries should be found as well. (3) Experimental verification by RT-PCR of the in silico cloning genes and further by cDNA sequencing. And then we use the three means as reference: (1) Web searching or in silico cloning of the genes of different species, especially mouse and rat homologous genes, and thus judging the gene existence by ontology. (2) By using the released genes in public domain as standard, which should be highly homologous to our verified genes, especially the released human genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS, we try to clone each a highly homologous complete gene similar to the released genes in public domain according to the strategy we developed in this paper. If we can not get it, our verified gene may be correct and the released gene in public domain may be wrong. (3) To find more evidence, we verified our cloned genes by RT-PCR or hybrid technique. Here we list some errors we found from NCBI GENOME ANNOTATION PROJECT REFSEQs: (1) Insert a base in the ORF by mistake which causes the frame shift of the coding amino acid. In detail, abase in the ORF of a gene is a redundant insertion, which causes a reading frame shift in the translation of an alternative protein, such as LOC124919 is wrong form of C17 orf32 (with mouse and rat orthologs determined by us). (2) Put together by mistake (with force). This is a wrong assembly of non-relating cDNA segment, such as LOC147007 is wrong form of C17orf32. (3) Mistakenly insert a base or one section of cDNA in the ORF which causes it ending beforehand, only coding cDNA sequence of N-terminal amino acids, incomplete. For example, LOC123722 is wrong form of SPRYD1, and even the human hypothetical gene LOC126250 or PDCD5 is wrong form of our PDCD5 (TFAR19). (4) Incomplete, only coding cDNA sequence of C-terminal amino acids. For example, human LOC149076 and mouse LOC230761 are wrong form of our verified human ZNF362 and mouse Zfp362, respectively. (5) Incomplete, only coding one section of coding protein cDNA sequence of correct gene ORF, lacking N-terminal and C-terminal amino acids sequence, and at the same time, mistakenly anticipates the first non-initiation codon amino acid of the incomplete protein amino acid as the initiation codon, e.g. anticipating L as M. For example, LOC200084 is wrong form of ZNF362. (6) Mistakenly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However,these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone,and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. The articles published in the past did not clearly point out the existence of mistakes in the NCBI human gene mode reference sequence. At the Seventh International Human Genome Conference held in April 2002, we first published the researching result on this aspect in the communication form of Posterly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However, these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone, and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗

PubMed2001

Anatomy of Escherichia coli ribosome binding sites.

During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon. The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking. Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits. To build the model, we first aligned the SD region by maximizing the information content there. The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts. This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern. We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model. A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites. Models were refined cyclically by removing non-conforming weak sites. After this procedure, models derived from either the original or the revised gene start annotation were similar. Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models. Such models should be useful for refining gene-start predictions of any sequenced bacterial genome.

Base Sequence↗

PubMed2001

Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium.

The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin (CN). Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher molecular mass protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5'RACE amplification of the smaller transcript yielded cDNAs lacking the 5' non-translated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5' untranslated region and at least the first AUG initiator codon, and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene.

5' Untranslated Regions↗

PubMed1987

The structure of the Ultrabithorax promoter of Drosophila melanogaster.

The sequence of 4118 nucleotides upstream of the putative initiator codon of the D. melanogaster Ultrabithorax protein was determined. The transcription initiation site for the corresponding mRNA was identified by S1 nuclease mapping and primed extension. It appears that all embryonic RNA products that encode the first protein exon are initiated approximately one kilobase upstream of the initiator methionine, suggesting a unique Ultrabithorax promoter. The unusually long mRNA leader is unspliced. Upstream of the initiator codon occur two additional methonine codons; the first one is followed by an open reading frame encoding a putative polypeptide of 69 amino acids. We discuss the role of the leader and 5' flanking sequences with respect to transcriptional and posttranscriptional control of Ultrabithorax gene expression.

Journal Article↗

PubMed1991

Immunological and virological characterization of improved construction of recombinant vaccinia virus expressing rinderpest virus hemagglutinin.

We constructed a recombinant vaccinia virus (RVV) expressing rinderpest virus (RPV) hemagglutinin (H) by modifying the promoter region of the original RVV. The promotor region was modified at three points, i.e., an outframe ATG was eliminated, the sequence between the promoter and initiation codon was shortened and the base sequence just upstream of the initiation codon was changed. As compared with the original RVV, the modified RVV was found to produce a remarkably large amount of H protein in infected rabbit kidney cells cultured in vitro and to induce high titers of anti-RPV-H antibodies in rabbits. The median protective doses in rabbits of the modified and of the original RVVs were 10(2) pfu and 10(3.5) pfu, respectively, indicating that the modified RVV was at least 10-times more effective in protection than the original. The neurovirulence of the modified RVV and the parental LC16mO strain was roughly at the same level, and was much lower than that of WR strain. The modified RVV was as heat-stable as the original one. These results indicate that the modified RVV could be a candidate rinderpest vaccine for further examinations including cattle.

Animals↗

PubMed1992

Viral spliced RNA are produced, encapsidated and reverse transcribed during in vivo woodchuck hepatitis virus infection.

By the use of reverse transcription followed by polymerase chain reaction (RT-PCR), we have identified one shorter than full-length, pregenomic viral RNA species in liver samples of woodchucks chronically infected with the woodchuck hepatitis virus (WHV). The spliced WHV RNA of about 2.4 kb in length was cloned and partially sequenced. The splicing donor and acceptor sites of this novel RNA are located, respectively, 130 nucleotides downstream of the ATG initiation codon of the core gene and 21 nucleotides upstream of the initiation codon of the pre-S2 surface gene. The splicing event generates a new core-polymerase fusion protein and removes the terminal protein domain and the spacer region of the polymerase gene. A nucleotide probe specific for the splice junction was used following RT-PCR, to further confirm the existence of this spliced RNA in the liver of seven WHV-infected woodchucks. Deleted viral DNA molecules corresponding to the 2.4 kb spliced RNA were also detected in the liver and, to a lesser extent, in the serum of infected woodchucks, suggesting that this spliced RNA can be encapsidated and reverse-transcribed during the course of natural WHV infection.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of the 5'-flanking sequence for the human DNA topoisomerase II beta gene.

Mammalian cells express two isoforms of type II DNA topoisomerase which are the intracellular targets of many structurally diverse antineoplastic agents. The levels of topoisomerase II isozymes are important determinants for the sensitivity of cells to the cytotoxicity of drugs that target topoisomerase II. To investigate whether the expression of topoisomerase II isoforms is coordinated and the mechanisms governing their expression in the context of drug resistance, the 5'-flanking sequence for the gene of human topoisomerase IIbeta isoform was cloned and characterized. The 5'-flanking region has a very high GC content and contains no canonical TATA box element. Two separate transcriptional start sites are located to an adenine and a guanine, 193 and 89 nucleotides, respectively, upstream from the ATG translation initiation codon. Except for a small region immediately upstream of the translation initiation codon, there is no obvious sequence homology between the 5'-flanking sequences of human topoisomerase IIbeta gene and the previously described alpha gene. Transient expression assays with different 5'- and 3'-deletions of the 5'-flanking region of the topoisomerase IIbeta gene have delineated regions important for transcriptional regulation of the gene. Interestingly, sequences within the first intron also contribute to the promoter activity. Gel mobility shift studies demonstrate that protein factors from the nuclear extracts can bind specifically to the downstream elements and may participate in transcriptional regulation.

Antigens, Neoplasm↗

PubMed2003

High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles.

The coat protein (CP) of Johnson grass mosaic virus (JGMV) auto-assembles to form virus-like particles (VLPs) and hence could be useful for presenting small peptides to the immune system. We are therefore attempting to synthesize JGMV CP in large amounts in Escherichia coli. The JGMV CP-encoding DNA, cloned under the bacteriophage T7 promoter, showed only low levels of CP synthesis in E. coli. The predicted secondary structure of the CP mRNA showed that its translational initiation codon was part of a stable hairpin-loop structure. The initiation codon could be relieved of the hairpin-loop structure by substitution of three neighboring nucleotides. This resulted in a single amino acid change at the N-terminus of the protein. The modified RNA translated very efficiently, resulting in at least 16-fold higher CP accumulation in E. coli. The N-terminal amino acid substitution did not affect CP folding, as it auto-assembled in E. coli to form VLPs.

Base Sequence↗

PubMed1992

Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.

Alternative Splicing↗

PubMed1995

Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus.

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.

Amino Acid Sequence↗

PubMed1985

Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12.

The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.

Amino Acid Sequence↗

PubMed1987

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.

Acyltransferases↗