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PubMed2004

Archaeal peptidyl prolyl cis-trans isomerases (PPIases) update 2004.

PPIases are ubiquitous in living organisms. While three families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases. Among 13 cyclophilins found in Archaea, only Halobacterium cyclophilin (HbsCyP19) has been characterized. This is a cyclosporin A (CsA) sensitive CyP with a molecular weight of 19.4 kDa. The PPIase activity and CsA sensitivity of HbsCyP19 is higher at higher salt concentration in the medium. No parvulin except a homolog in Cenarchaeum symbiosum has been found in Archaea. Two types of FKBPs, 26-30 kDa long-type and 17-18 kDa short-type FKBP, have been found in Archaea. Up to date, 12 short-type FKBPs and 18 long-type FKBPs have been known. The short-type FKBPs and N-terminal sequences of the long-type FKBPs are similar to each other and show homology to human FKBP12 (HsFKBP12). However, they have two insertion sequences in the regions corresponding to bulge and flap loops of HsFKBP12. The long-type archaeal FKBPs have additional ca. 100 amino-acid sequences at their C-terminal regions. A short-type archaeal FKBP from Methanothermococcus thermolithotrophicus has not only a PPIase activity but also a chaperone-like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates. Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the flap seemed to be important. Three-dimensional structure of this FKBP showed that the insertion in the flap makes a domain which has a hydrophobic surface. Coexpression of aggregation prone proteins with these archaeal FKBPs were shown to improve their expression in soluble fraction in Escherichia coli. Fusion protein of the archaeal FKBP and an aggregation prone protein also show improved expression of the latter in E. coli.

Amino Acid Sequence↗
PubMed1995

Type 3 lodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme.

Type 3 iodothyronine deiodinase (D3) catalyzes the conversion of T4 and T3 to inactive metabolites. It is highly expressed in placenta and thus can regulate circulating fetal thyroid hormone concentrations throughout gestation. We have cloned and expressed a 2.1-kb human placental D3 cDNA which encodes a 32-kD protein with a Km of 1.2 nM for 5 deiodination of T3 and 340 nM for 5' deiodination of reverse T3. The reaction requires DTT and is not inhibited by 6n-propylthiouracil. We quantitated transiently expressed D3 by specifically labeling the protein with bromoacetyl [125I]T3. The Kcat/Km ratio for 5 deiodination of T3 was over 1,000-fold that for 5' deiodination of reverse T3. Human D3 is a selenoenzyme as evidenced by (a) the presence of an in frame UGA codon at position 144, (b) the synthesis of a 32-kD 75Se-labeled protein in D3 cDNA transfected cells, and (c) the presence of a selenocysteine insertion sequence element in the 3' untranslated region of the mRNA which is required for its expression. The D3 selenocysteine insertion sequence element is more potent than that in the type 1 deiodinase or glutathione peroxidase gene, suggesting a high priority for selenocysteine incorporation into this enzyme. The conservation of this enzyme from Xenopus laevis tadpoles to humans implies an essential role for regulation of thyroid hormone inactivation during embryological development.

Amino Acid Sequence↗
PubMed1997

The histone H1 genes of the dipteran insect, Chironomus thummi, fall under two divergent classes and encode proteins with distinct intranuclear distribution and potentially different functions.

Four histone H1 genes of the midge, Chironomus thummi piger, and three H1 genes of the subspecies C. thummi thummi have been cloned and assigned to the four different H1 proteins from C. thummi larvae. Together with an earlier cloned H1 gene from C. thummi thummi [Hankeln, T. & Schmidt, E. R. (1991) Chromosoma 101, 25-31], these genes probably constitute the complete complement of H1 genes in both subspecies. They were found to fall under two classes that differ remarkably in their gene copy numbers, genomic organization, structure of flanking sequences, codon usage, and expression during embryonic development, and that encode H1 proteins of divergent structure. Histone H1 I-1 contains an inserted sequence, KAPKAPKAPKSPKAE in C. thummi piger, and KAPKAPKSPKAE in C. thummi thummi, that is lacking in the other H1 variants, H1 II-1, H1 II-2, and H1 III-1. In the immediate neighbourhood to the inserted sequence, a substitution in the H1 I-1 protein sequence dramatically enhances the potential to form a reversed turn. In early development, H1 I-1 is expressed at a higher rate than the other H1 genes. The transcripts have a size of about 1 kb; in addition, the H1 I-1 gene exhibited two minor transcripts of about 2.5 and > 3 kb size in middle blastoderm that are possibly polyadenylated. Together with our earlier finding that histone H1 I-1 is found in a limited number of polytene chromosome bands whereas the other H1 histones are uniformly distributed in chromatin, these results intimate functional differences between the two classes of H1 genes and their products.

Amino Acid Sequence↗
PubMed1997

The evolution of an intron: analysis of a long, deletion-prone intron in the human dystrophin gene.

The sequence of a 112-kb region of the human dystrophin (DMD/BMD) gene encompassing the deletion prone intron 7 (110 kb) and the much shorter intron 8 (1.1 kb) has been determined. Recognizable insertion sequences account for approximately 40% of intron 7. LINE-1 and THE-1/LTR sequences occur in intron 7 with significantly higher frequency than would be expected statistically while Alu sequences are underrepresented. Intron 7 also contains numerous mammalian-wide interspersed repeats, a diverse range of medium reiteration repeats of unknown origin, and a sequence derived from a mariner transposon. By contrast, the shorter intron 8 contains no detectable insertion sequences. Dating of the LI and Alu sequences suggests that intron 7 has approximately doubled in size within the past 130 million years, and comparison with the corresponding intron from the pufferfish (Fugu rubripes) suggests that the intron has expanded some 44-fold over a period of 400 million years. The possible contribution of the insertion elements to the instability of intron 7 is discussed.

Base Composition↗
PubMed2002

A third transposable element, ISPpu12, from the toluene-xylene catabolic plasmid pWW0 of Pseudomonas putida mt-2.

A 3,372-bp insertion sequence, ISPpu12, has been identified on the archetypal toluene-xylene TOL catabolic plasmid pWW0 from Pseudomonas putida mt-2. The insertion sequence element is located on the plasmid between bases 84397 and 87768 in a region which also contains the termini and transposase genes of the catabolic transposons Tn4651 and Tn4653 (A. Greated, L. Lambertson, P. A. Williams, and C. M. Thomas, Environ. Microbiol., in press). ISPpu12 has terminal inverted repeats of 24 bp with three mismatches and contains four open reading frames, a tnpA homologue and three open reading frames (lspA, orf1, and orf2) of undetermined function. After insertion in vitro of a Km(r) cassette into ISPpu12 either in the intergenic region between orf1 and orf2 or directly into the orf1 gene and ligation into a suicide vector, the modified ISPpu12-Km transposes at high frequency, often in multiple copies, into the chromosome of a P. putida recipient. Inactivation of lspA, orf1, and orf2 by introducing a 7-bp deletion into the 5' region of each gene had no major effect upon transposition, but a similar mutation of tnpA completely eliminated transposition. Analysis of the literature and of strains derived from the chlorobenzoate-degrading Pseudomonas sp. strain B13 suggests that the promiscuity of this element has played an important role in the history of plasmid pWW0. Database comparisons and the accompanying paper (A. J. Weightman, A. W. Topping, K. E. Hill, L. L. Lee, K. Sakai, J. H. Slater, and A. W. Thomas, J. Bacteriol. 184:6581-6591, 2002) show that ISPpu12 is a transposable element also found in other bacteria.

Bacterial Proteins↗
PubMed1998

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue.

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.

Amino Acid Sequence↗
PubMed2001

Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality.

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.

Algorithms↗
PubMed2005

Characterization of 954 bovine full-CDS cDNA sequences.

BACKGROUND: Genome assemblies rely on the existence of transcript sequence to stitch together contigs, verify assembly of whole genome shotgun reads, and annotate genes. Functional genomics studies also rely on transcript sequence to create expression microarrays or interpret digital tag data produced by methods such as Serial Analysis of Gene Expression (SAGE). Transcript sequence can be predicted based on reconstruction from overlapping expressed sequence tags (EST) that are obtained by single-pass sequencing of random cDNA clones, but these reconstructions are prone to errors caused by alternative splice forms, transcripts from gene families with related sequences, and expressed pseudogenes. These errors confound genome assembly and annotation. The most useful transcript sequences are derived by complete insert sequencing of clones containing the entire length, or at least the full protein coding sequence (CDS) portion, of the source mRNA. While the bovine genome sequencing initiative is nearing completion, there is currently a paucity of bovine full-CDS mRNA and protein sequence data to support bovine genome assembly and functional genomics studies. Consequently, the production of high-quality bovine full-CDS cDNA sequences will enhance the bovine genome assembly and functional studies of bovine genes and gene products. The goal of this investigation was to identify and characterize the full-CDS sequences of bovine transcripts from clones identified in non-full-length enriched cDNA libraries. In contrast to several recent full-length cDNA investigations, these full-CDS cDNAs were selected, sequenced, and annotated without the benefit of the target organism's genomic sequence, by using comparison of bovine EST sequence to existing human mRNA to identify likely full-CDS clones for full-length insert cDNA (FLIC) sequencing. RESULTS: The predicted bovine protein lengths, 5' UTR lengths, and Kozak consensus sequences from 954 bovine FLIC sequences (bFLICs; average length 1713 nt, representing 762 distinct loci) are all consistent with previously sequenced mammalian full-length transcripts. CONCLUSION: In most cases, the bFLICs span the entire CDS of the genes, providing the basis for creating predicted bovine protein sequences to support proteomics and comparative evolutionary research as well as functional genomics and genome annotation. The results demonstrate the utility of the comparative approach in obtaining predicted protein sequences in other species.

5' Untranslated Regions↗
PubMed1990

Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein E1.

Genomic RNA of hog cholera virus (HCV) strain Brescia was cloned and sequenced. The nucleotide sequence was deduced from overlapping cDNA clones and comprises 12,283 nucleotides. We cloned the complete 3' end of the HCV genome, but could not unequivocally prove that the cDNA sequence also completely covers HCV RNA at the 5' end. The HCV genome contained one large open reading frame, which spans the viral plus strand RNA and encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438,300. To identify structural HCV glycoproteins, we prepared rabbit antisera against three synthetic peptides deduced from the sequence. Because one of these antisera reacted with a 51- to 54-kDa glycoprotein (envelope protein E1 of HCV) on Western blot, the genomic position of the sequence encoding gp51-54 could be clearly established. The amino acid sequence of Brescia was compared with that of HCV strain Alfort and that of BVDV strains NADL and Osloss. The degree of homology between the two HCV strains was 93%, and between Brescia and the BVDV strains about 70%. NADL contained an inserted sequence of 90 amino acids that was absent from the sequences of Brescia, Alfort, and Osloss, whereas Osloss contained an inserted sequence of 76 amino acids that was absent from the sequences of Brescia, Alfort, and NADL. Sequences in p80, the most homologous protein among pestiviruses, showed similarity to six sequence motifs found conserved in helicase-like proteins represented by eIF-4A. Furthermore, a trypsin-like serine protease domain detected in p80 of BVDV was also found conserved in HCV, suggesting that pestivirus p80 may be bifunctional.

Amino Acid Sequence↗
PubMed2003

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Base Sequence↗
PubMedDate not supplied

Transposition in prokaryotes: transposon Tn501.

Bacteria contain a large number of transposable elements that can be categorized in four major groups according to their mechanisms of transposition. These are: class I: insertion sequences (IS) and compound transposons (with IS sequences at their termini) which usually require only one protein for transposition to occur (e.g. Tn10); class II: complex transposons and insertion sequences with short inverted repeats in which transposition is replicative and requires two gene products (e.g. Tn3); class III: transposable bacteriophage (e.g. Mu). The fourth group consists of the transposons and IS of variable mechanism, which do not fall into the above classes (e.g. Tn7). We have studied the mechanism of transposition of Tn501 and Tn21, closely-related class II mercury-resistance transposons, which transpose via a cointegrate intermediate. By using genetic methods, we have shown that the region of the 989 amino acid transposase between amino acids 57 and 186 determines the specificity for recognition of the 38-bp terminal inverted repeats of the transposon in normal transposition and for replicon fusion catalysed by a single transposon terminus. The Tn501 transposase has been over-expressed and is functional in vivo, raising the frequency of transposition approximately 10(4)-fold.

Amino Acid Sequence↗
PubMed1998

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Amino Acid Sequence↗
PubMed1996

Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.

Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.

Amino Acid Sequence↗
PubMed2000

Archaeal peptidyl prolyl cis-trans isomerases (PPIases).

PPIases are ubiquitous in living organisms. While 3 families of PPIases, cyclophilin (CyP), FK506 binding protein (FKBP) and parvulin (Pvn), have been studied in detail in Eukarya and Bacteria (eubacteria), little is known about archaeal PPIases. Among 2 cyclophilins found in Archaea, only Halobacterium cyclophilin (HcCyP19) has been characterized. It is a cyclosporin A (CsA) sensitive CyP with a MW of 19.4kDa. The PPIase activity and CsA sensitivity of this CyP is higher at higher salt concentration in the medium. No parvulin or its homolog has been found in Archaea. Two types of FKBPs, 26-30kDa long type and 17-18 kDa short type FKBP, have been found in Archaea. While the N-terminal regions of these 2 type FKBPs are similar to each other, the long type archaeal FKBP has an additional ca. 100 amino-acid sequence at its C-terminal region. In comparison with human HsFKBP12, the N-terminal region of the archaeal FKBP has 2 insertion sequences in the regions corresponding to Bulge and Flap of HsFKBP12. A short type archaeal FKBP from Methanococcus thermolithotrophicus has been shown to have not only a PPIase activity but also a chperone like activity, which includes protein refolding and aggregation suppressing activities with regard to protein folding intermediates. Mutational analysis revealed that this chaperone-like activity was independent of the PPIase activity, and that the insertion sequence in the region corresponding to the Flap seemed to be important.

Amino Acid Sequence↗
PubMed1988

Impairment of yeast pre-mRNA splicing by potential secondary structure-forming sequences near the conserved branchpoint sequence.

The absolutely conserved TACTAAC box within introns of RNA polymerase II-transcribed genes of the yeast Saccharomyces cerevisiae serves an indispensable role in lariat formation. We show in this report that rather short palindromic sequences inserted into the yeast actin gene intron immediately 3' to the TACTAAC box block the second but not the first splicing step. In contrast, a palindromic sequence inserted some 23 bp 3' of the TACTAAC box did not affect correct and efficient splicing. The data suggest that hairpin structures that might form adjacent to the branchsite sequence interfere with some necessary alteration of the spliceosome required for 3' intron cleavage and exon ligation.

Actins↗
PubMed1996

Molecular characterisation of adenosylhomocysteinase from Trichomonas vaginalis.

The enzyme S-adenosylhomocysteine hydrolase (SAHH) has been identified as a potential target for chemotherapy in protozoan parasites including Trichomonas vaginalis. To investigate this area of trichomonad metabolism in more detail, we have isolated and characterised a gene which encodes this activity from the WAA38 strain of this parasite. The gene was isolated by probing a Bg/II genomic mini-library with a fragment of the gene generated by thermal cycling using degenerate oligonucleotide primers. A 5.9-kb Bg/II clone was isolated and has been partially sequenced to reveal a 1458-bp open reading frame which encodes a 486-residue polypeptide (computed molecular mass of 53.4 kDa). The deduced amino acid sequence showed a high degree of sequence similarity to the hydrolases from other species, but was most similar to the enzyme from photosynthetic organisms. The trichomonal sahh gene also contains two "insertion sequences', one of which appears to be unique to this parasite while the second has previously been found only in photosynthetic organisms and in Plasmodium falciparum. Characterisation of the sahh mRNA from T. vaginalis confirmed that both of these insertion sequences (encoding 9 and 37 amino acid residues, respectively) are expressed in the protein product. The sahh mRNA is similar to those characterised from other protozoa in having a short, 12-bp untranslated 5'-leader sequence but the leader sequence does not conform well with the consensus sequence of the other mRNAs. Finally, Southern blots and sequence differences between genomic and cDNA clones indicate that there are multiple copies of the sahh gene in T. vaginalis.

Adenosylhomocysteinase↗
PubMed1991

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.

Amino Acid Sequence↗