Showing papers on "Sequence analysis published in 1988"
TL;DR: The efficacy of this cDNA cloning strategy was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases.
Abstract: We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.
4,673 citations
TL;DR: Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis.
2,636 citations
TL;DR: DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers, designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work.
Abstract: DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.
984 citations
TL;DR: Northern blot analysis suggests that the new members of protein kinase C exist in the brain and some other tissues.
666 citations
TL;DR: Cl cloning of the complete coding sequence of the human androgen receptor (hAR) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%.
Abstract: Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.
570 citations
TL;DR: Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene, which will now facilitate epitope mapping studies and shows a high degree of homology with animal and plant cysteine proteases.
Abstract: A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.
493 citations
TL;DR: Evidence is presented from DNA sequence analysis that HCMV encodes a molecule similar to the MHC class-I antigens of higher eucaryotes, and it is proposed that this protein is responsible for the observed β2-m binding.
Abstract: Primary infection with human cytomegalovirus (HCMV) is persistent and widespread, with symptoms that are mostly subclinical but can cause serious illness or death, particularly in immunosuppressed patients1,2. Recently3, proteins from HCMV were shown to bind β2-microglobulin (β2-m) a protein that is normally found associated with the class-I major histocompatibility complex (MHC) antigens, which are essential for self-non-self recognition in the immune response4. These findings led to the proposal that the virus may use β2-m binding as an infection mechanism5. Here we present evidence from DNA sequence analysis that HCMV encodes a molecule similar to the MHC class-I antigens of higher eucaryotes, and propose that this protein is responsible for the observed β2-m binding. The deduced amino-acid sequence of the HCMV class-I-like protein reveals conservation of typical features of class-I structure, but we predict that the gene is not spliced, in contrast to the cellular genes.
479 citations
Journal Article•
TL;DR: The complete sequence of P30, the major surface Ag of the protozoan parasite, Toxoplasma gondii, has been deduced through the cloning and analysis of its gene and the apparent 5' and 3' ends of the P30 mRNA transcript are identified.
Abstract: The complete sequence of P30, the major surface Ag of the protozoan parasite, Toxoplasma gondii, has been deduced through the cloning and analysis of its gene. Using polyclonal serum specific for P30, we have isolated a P30 cDNA clone from a lambda gt11 cDNA expression library derived from tachyzoites of T. gondii (RH strain). This clone produces a beta-galactosidase fusion protein which reacts with several anti-P30 mAb. In addition, polyclonal anti-serum raised to the fusion protein reacts with purified P30 protein and exclusively with P30 in a whole cell lysate of T. gondii. This cDNA clone was used to isolate near full-length cDNA molecules and a cosmid clone containing the P30 gene. Sequence analysis of the cDNA reveals a single open reading frame with coding capacity for 34.7 kDa of primary translation product (consistent with the apparent Mr of P30 on SDS-acrylamide gels) including a presumptive hydrophobic signal sequence. The P30 primary translation product also has a carboxy-terminal hydrophobic tail which is predictive of a posttranslational cleavage and modification with a glycolipid anchor. We have identified the apparent 5' and 3' ends of the P30 mRNA transcript which is extremely abundant, 1500 nucleotides in length, and polyadenylated. The P30 gene is single copy and contains no introns.
459 citations
TL;DR: Evidence is provided that a cDNA that encodes a major part of the human androgen receptor is isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to parts of the region encoding the DNA-binding domain of steroid receptors.
368 citations
TL;DR: It is concluded that gamma E evolved into gamma F in stem-simians and an elevated non-synonymous substitution rate characterizes this transition.
354 citations
TL;DR: Two 35-kDa proteins from rat peritoneal lavages that inhibit phospholipase A2 activity are purified and it is confirmed that they are lipocortin-related, and the two inhibitors are referred to aslipocortins III and V.
TL;DR: The complete amino acid sequence of a third sodium channel (designated sodium channel III) from rat brain has been deduced by cloning and sequence analysis of the cDNA.
TL;DR: By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain, and was absent in other species of the genus Listeria.
Abstract: To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.
TL;DR: A subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication is developed, used to isolate three transcripts whose abundance is increased in scrapie-infected brain.
Abstract: We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in pi H3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapie-infected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of alpha-crystallin; the latter two may represent a response to stress.
TL;DR: It is shown that CHI and CHS genes are coordinately and tissue‐specifically expressed in a developmental and light‐regulated manner and coordinate induction of both mRNAs is observed after continuous irradiation of Petunia plantlets with UV light, probably as part of the plants UV defence mechanism.
Abstract: In this paper we report the isolation of cDNA clones encoding the flavonoid-biosynthetic enzyme chalcone flavanone isomerase (CHI) from Petunia hybrida. A nearly full size cDNA clone, isolated from a corolla-specific expression library, was characterized by sequence analysis. Using this CHI cDNA and the previously cloned flavonoid-specific chalcone synthase (CHS) cDNA we show that CHI and CHS genes are coordinately and tissue-specifically expressed in a developmental and light-regulated manner. Furthermore, coordinate induction of both mRNAs is observed after continuous irradiation of Petunia plantlets with UV light, probably as part of the plants UV defence mechanism. The two CHI genes, denoted A and B, were isolated from a genomic library of the Petunia inbred line V30. Both genes are transcriptionally active: gene A is transcribed in corolla, tube and UV-irradiated plantlets (1.0 kb mRNA), whereas gene B is only transcribed in immature anthers (1.0 kb mRNA). In combination with Southern blot analysis these data implicate the presence of two distinct non-allelic CHI genes in the genome of the P. hybrida line V30. Unexpectedly, mature anthers accumulate a 0.3 kb larger CHI RNA. This RNA is transcribed from CHI gene A and has a 0.3 kb 5' extension relative to the gene A transcript found in corolla tissue. Furthermore it is neither coordinately expressed with CHS mRNA nor UV inducible. Its biological function is still obscure, since no active CHI enzyme could be demonstrated in the same tissue.
TL;DR: In this paper, a set of cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerases I serum.
Abstract: cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.
TL;DR: A method for screening and comparing large numbers of unselected integration events for cell target sequences in retrovirus replication and reveals the presence of a number of strongly preferred integration targets.
TL;DR: A Drosophila gene that encodes neuropeptides related to molluscan Phe-Met-Arg-Phe-NH2 (FMRFamide) was isolated by screening a genomic library with a fragment of an Aplysia phe-met-argument-phe-nh2 cDNA and with synthetic oligonucleotides.
Abstract: A Drosophila gene that encodes neuropeptides related to molluscan Phe-Met-Arg-Phe-NH2 (FMRFamide) was isolated by screening a genomic library with a fragment of an Aplysia Phe-Met-Arg-Phe-NH2 cDNA and with synthetic oligonucleotides. This gene was used to isolate a cDNA from a Drosophila adult head cDNA library. The cDNA was defined by sequence analysis to encode 13 peptides that have Phe-Met-Arg-Phe-NH2 or related sequences at their carboxyl termini. Other putative neuropeptides, including one that has homology to mammalian corticotropin-releasing factor, are present in the deduced approximately equal to 39-kDa precursor. Southern blot analysis suggested the presence of a single Phe-Met-Arg-Phe-NH2-like gene within the haploid genome. RNA blot analysis indicated the expression of at least two transcripts of approximately equal to 1.7 and approximately equal to 0.7 kilobases. Both transcripts are evident throughout larval, pupal, and adult developmental stages. In situ hybridization was used to localize this neuropeptide gene to band 46C on the right arm of the 2nd chromosome. These data provide the basis for utilizing the advanced genetics and molecular techniques of Drosophila to address complex aspects of neuropeptide expression and function.
TL;DR: Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation.
Abstract: Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c-locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.
TL;DR: The results of this analysis indicate that predisposition to PV is conferred by different sequences in DR4 and DRw6 haplotypes.
Abstract: The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the HLA serotypes DR4 and DRw6. Susceptibility to PV could be conferred either by sequences shared between the DR4 and DRw6 haplotypes or by different sequences in these haplotypes. We have examined the distribution of DR and DQ beta-chain and DQ alpha-chain alleles in PV patients and in control subjects by hybridization with oligonucleotide probes and sequence analysis of in vitro amplified DNA. Ninety percent (34/38) of the DR4 haplotypes in patients contain a specific DR beta I sequence present in 36% (16 of 44) of DR4 controls (P = 0.001). This sequence is also found in DRw6 haplotypes. However, it is present in only 25% (6 of 24) of DRw6 patients. The results of our analysis indicate that predisposition to PV is conferred by different sequences in DR4 and DRw6 haplotypes. The DR4 susceptibility is highly associated with the Dw10 DR beta I allele, implicating the polymorphic residues in the third hypervariable region. The DRw6 susceptibility is strongly associated with a rare DQ beta allele (DQB1.3). This allele differs from a common DQ beta allele (DQB1.1) only by a valine----aspartic acid substitution at position 57.
TL;DR: It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.
Abstract: Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from coumaroyl-CoA and malonyl-CoA. Two cDNA clones, pGSC1 and pGSC2, have been isolated from cDNA libraries established with poly(A)-rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSG10 contains the 5' and pGSG11 the 3' end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a common scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.
TL;DR: The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA and the sequence analysis is presented.
Abstract: The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA. The phosphorothioate groups are incorporated into the nucleic acid in four separate enzymatic reactions, with three of the natural nucleoside triphosphates and one alpha-thiotriphosphate in each reaction. Selective strand cleavage is achieved through alkylation to form the hydrolytically labile phosphorothioate triester. As an example, the sequence analysis is presented of M13 phage DNA and of RNA prepared by transcription with SP6 RNA polymerase.
TL;DR: The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA by automated Edman degradation.
TL;DR: Alignment of the 15 domains and the extracytoplasmic domain of the cation-dependent mannose 6-phosphate receptor shows that all have sequence similarities and suggests that all are homologous.
01 Jan 1988
TL;DR: Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher numbers of transcripts for TGF-beta 1 than for T GF-beta 2, although they produce more TGF,beta 2 protein than TGF -beta 1, suggesting that there is a post-transcriptional level of regulation for the production of these proteins.
Abstract: We have obtained a cDNA clone coding for human transforming growth factor (TGF)-β2. The clone was isolated from a tamoxifen-treated human prostatic adenocarcinoma cell line (PC-3) using oligonucleotide probes based on the partial amino acid sequence of purified TGF-β2. The cDNA sequence predicts that TGF-β2 is synthesized as a 442-amino-acid polypeptide precursor from which the mature 112-amino-acid TGF-β2 subunit is derived by proteolytic cleavage. The proteins coded for by the human TGF-β1 and TGF-β2 cDNAs show an overall homology of 41%. The mature and amino-terminal precursor regions show 71% and 31% homology, respectively. Northern blot analysis identified TGF-β2 transcripts of 4.1, 5.1, and 6.5 kb using mRNA from several different sources. Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher numbers of transcripts for TGF-β1 than for TGF-β2, although they produce more TGF-β2 protein than TGF-β1. This suggests that there is a post-transcript...
TL;DR: A comparative sequence analysis of the 5' noncoding region of a subgroup of the picornavirus, including the polioviruses, coxsackie B3, and the human rhinovirus, reveals the conservation of certain features despite the divergence of sequence, implicates a biologically functional role for this region.
TL;DR: Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and this sequence is tentatively designated Gx alpha.
Abstract: We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.
TL;DR: The discovery of a family of transposable elements of the crucifer Arabidopsis thaliana, which are referred to as Ta1 elements, shows that it is closely related to retrotransposons and integrated retroviral proviruses, and has transposed in the period of time since divergence of two races of A.Thaliana.
Abstract: The fast generation time, small genome size and extensive genetic map of the crucifer Arabidopsis thaliana have made it the subject of an increasing number of studies in plant molecular genetics. As transposable elements have greatly facilitated genetic analysis in a variety of species, we have attempted to identify an endogenous A. thaliana transposable element. We report here the discovery of a family of such elements, which we refer to as Ta1 elements. Sequence analysis of one such element shows that it is closely related to retrotransposons and integrated retroviral proviruses, being bound by a direct sequence repeat and having an open reading frame with clear sequence similarity to the polyprotein of the Drosophila melanogaster retrotransposon copia. The sequence of an empty target site of a Ta1 element shows that insertion is accompanied by a five-base-pair target-site duplication and that Ta1 has transposed in the period of time since divergence of two races of A. thaliana.
TL;DR: Evidence is presented indicating that gene X and secA are cotranscribed, essential for protein export in Escherichia coli, and that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein.
Abstract: TheDNA sequence ofthesecAgene, essential forprotein export inEscherichia coli, wasdetermined and found toencode ahydrophic protein of901aminoacid residues with apredicted molecular weight of101,902, consistent withitspreviously determined size andsubcelular location. Sequence analysis of9secA(Ts) mutations conferring general protein export andsecAregulatory defects revealed that these mutations were clustered inthree specific regions within thefirst 170aminoacidresidues oftheSecAprotein andwerethe result ofsingle aminoacidchanges predicted tobeseverely disruptive ofprotein structure andfunction. The DNA sequence immdtelyupstream ofsecAwasshowntoencode apreviously inferred gene, geneX. Sequence analysis ofa conditionally lethal ambermutation, amlO9,previously inferred tobelocated proximally inthesecAgene, revealed that itwaslocated distally ingeneXandwasconditionaly lethal dueto its polar effect onsecAexpression. Thisandadditional evidence arepresented indicating that geneXandsecA arecotranscribed. Biochemical andgenetic approaches havebeenusedto study themolecular mechanisms responsible forprotein localization inprocaryotic andeucaryotic cells. Suchstudies reveal that protein translocation across thebacterial plasma membrane andtheeucaryotic roughendoplasmic reticular membrane occurbyapparently homologous andconserved mechanisms. Sharedfeatures include similar signal sequences andstoptransfer sequences onpresecretory protein precursors that arerequired fortheir correct localization and topology (46), frequent removal ofamino-terminal signal peptides bysignal peptidases ofsimilar specificity (45), a dependence onanenergy source suchasATPorelectrochemical membrane potential formembrane translocation (6,13,14),anda requirement forsometypeofexport machinery (29, 35,44).
TL;DR: DNA sequence analysis of RpII215, the gene that encodes the Mr215,000 subunit of RNA polymerase II in Drosophila melanogaster, reveals that the 3'-terminal exon includes a region encoding a C-terminal domain composed of 42 repeats of a seven-residue amino acid consensus sequence.
Abstract: DNA sequence analysis of RpII215, the gene that encodes the Mr215,000 subunit of RNA polymerase II (EC 2.7.7.6) in Drosophila melanogaster, reveals that the 3'-terminal exon includes a region encoding a C-terminal domain composed of 42 repeats of a seven-residue amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A hemi- and homozygous lethal P-element insertion into the coding sequence of this domain causes premature translation termination and therefore truncation of the protein, leaving only 20 heptamer repeats. While loss of approximately 50% of the repeat structure in this mutant is a lethal event in vivo, enzyme containing the truncated subunit remains capable of accurate initiation at promoters in vitro. Moreover, treatment of purified intact RNA polymerase II with protease, to remove the entire repeat domain, does not eliminate the enzyme's ability to initiate accurately in vitro. Possible in vivo functions for this unusual protein domain are considered in light of these results.