Showing papers in "Nucleic Acids Research in 1983"
TL;DR: A procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters, including tRNA and Ad 2 VA, is developed.
Abstract: We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).
10,800 citations
TL;DR: A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations.
Abstract: The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.
2,101 citations
TL;DR: These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used, however, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler.
Abstract: We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.
1,049 citations
TL;DR: The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin gene.
Abstract: The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other actin genes, the beta-actin gene lacks the codon for Cys between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.
1,000 citations
TL;DR: Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasm or normal tissues, which might reflect an involvement of extensive demethylation of DNA in tumor progression.
Abstract: The over-all 5-methylcytosine (m5C) content of DNA from normal tissues varies considerably in a tissue-specific manner. By high-performance liquid chromatography, we have examined the m5C contents of enzymatic digests of DNA from 103 human tumors including benign, primary malignant and secondary malignant neoplasms. The diversity and large number of these tumor samples allowed us to compare the range of DNA methylation levels from neoplastic tissues to that of normal tissues from humans. Most of the metastatic neoplasms had significantly lower genomic m5C contents than did most of the benign neoplasms or normal tissues. The percentage of primary malignancies with hypomethylated DNA was intermediate between those of metastases and benign neoplasms. These findings might reflect an involvement of extensive demethylation of DNA in tumor progression. Such demethylation could be a source of the continually generated cellular diversity associated with cancer.
837 citations
TL;DR: Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions, and the effect was even more pronounced in transfections with linear forms of polyomas DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.
Abstract: Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. Exposure to chloroquine increased the proportion of transfected mouse cells to approximately 40%. From a culture of one million such cells, microgram quantities of newly synthesized viral DNA could be isolated. Similarly, the transformation frequency of rat cells following polyoma DNA transfection was approximately 6-fold increased by chloroquine treatment. The effect of the compound was even more pronounced in transfections with linear forms of polyoma DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.
630 citations
TL;DR: This work has constructed and tested several new vectors for P element-mediated gene transfer and describes a genomic sequence arrangement that apparently arose by the transposition of a 54 kb composite P element from a tetramer plasmid.
Abstract: We have constructed and tested several new vectors for P element-mediated gene transfer. These vectors contain restriction sites for cloning a wide variety of DNA fragments within a small, non-autonomous P element and can be used to efficiently transduce microinjected DNA sequences into the germ line chromosomes of D. melanogaster. The P element in one vector also carries the rosy gene which serves as an easily scored marker to facilitate the transfer of DNA fragments that do not themselves confer a recognizable phenotype. The failure of certain P element constructs to function as vectors suggests that P element sequences, in addition to the 31 bp inverse terminal repeats, are required in cis for transposition. Moreover, removal of the first 38 bp of the autonomous 2.9 kb P element appears to destroy its ability to provide a trans-acting factor (s) required for the transposition of non-autonomous P elements. Finally, we describe a genomic sequence arrangement that apparently arose by the transposition of a 54 kb composite P element from a tetramer plasmid.
482 citations
TL;DR: The complete nucleotide sequences of two different subtypes (adr and adw) of hepatitis B virus DNA cloned in E. coli were determined and Heterogeneity of the HBV genome in the clones with the same subtype was observed.
Abstract: The complete nucleotide sequences of two different subtypes (adr and adw) of hepatitis B virus (HBV) DNA cloned in E coli were determined The sequence of the viral genome of the adr clone was 3188 nucleotides long, and that of the adw clone was 3200 nucleotides long The adr and adw clones differed from the reported cloned ayw HBV DNA (3182 nucleotides long) in 112% and 100% of nucleotides, respectively Heterogeneity of the HBV genome in the clones with the same subtype was observed
416 citations
TL;DR: The strategy used to determine the sequence produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues.
Abstract: We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37. This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts of other transcription units. The strategy used to determine the sequence also produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues.
391 citations
TL;DR: The subtilisin gene from B. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pBS42, in Bacillus subtilis I-168 (Marburg strain), and a sequence of unknown function coding for roughly 75 amino acids between the signal sequence and the mature enzyme is proposed.
Abstract: The subtilisin gene from B. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pBS42, in Bacillus subtilis I-168 (Marburg strain). Greater than 95 percent of the expressed protease activity is secreted, and the activity is sensitive to inhibition by phenylmethylsulfonyl fluoride as expected for subtilisin. Bacillus subtilis transformants carrying the Bacillus amyloliquefaciens subtilisin gene in pBS42 (called pS4) secreted large amounts of a protein not seen in control pBS42 transformants. This protein migrated in SDS gels near the position of authentic subtilisin. The complete nucleotide sequence of the cloned gene has been determined using dideoxy sequencing methods. Ba131 exonuclease digestion studies at the 5' end of the gene have defined a 31 base pair stretch necessary for efficient expression of subtilisin. In addition to this putative promoter region, sequences have been assigned for ribosome binding, translation initiation, a signal peptide, the mature enzyme, and translation and transcription termination. A most interesting feature of the gene is a sequence of unknown function coding for roughly 75 amino acids between the signal sequence and the mature enzyme. It is proposed that this region serve as a pro-peptide as is commonly found in eukaryotic secreted proteases.
386 citations
TL;DR: The genetic distance from Xp110 to Xp223 was found to be approximately 40 centimorgans (cM), which provides experimental confirmation that 1cM corresponds to approximately 1,000 kilobase pairs of DNA for this region of the human X chromosome.
Abstract: The inheritance of two restriction fragment length polymorphisms (RFLPs) on the short arm of the human X chromosome has been studied relative to Duchenne muscular dystrophy. This provides a partial genetic map of the short arm of the human X chromosome between Xp110 and Xp223. The data were derived from the segregation between a RFLP located at Xp21-Xp223, the DMD locus, and a RFLP located at Xp110-Xp113. The genetic distance from Xp110 to Xp223 was found to be approximately 40 centimorgans (cM). This provides experimental confirmation that 1cM corresponds to approximately 1,000 kilobase pairs of DNA for this region of the human X chromosome. Our data confirm that the DMD mutation lies between Xp223 and Xp110. The availability of flanking probes surrounding the DMD locus will assist in the ordering of further DNA sequences relative to the mutation.
TL;DR: The results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels, and expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.
Abstract: We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa cells, involving the SV40 early promoter. Stable transformation efficiencies increase to 4% and 10% in HeLa S3 and monkey kidney CV-1 cells, respectively. Finally, expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.
TL;DR: From these data, it is estimated that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expression in the brain.
Abstract: 191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.
TL;DR: A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes and was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.
Abstract: A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.
TL;DR: The nucleotide sequence of the chick beta-actin gene was determined and it was found that the gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein.
Abstract: The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.
TL;DR: Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.
Abstract: Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.
TL;DR: A family of repeated restriction fragments whose molecular organization is apparently specific to the human X chromosome is identified and characterization and it is estimated that there are 5,000-7,500 copies of the 2.0 kb BamHI repeat per haploid genome.
Abstract: We report the identification and characterization of a family of repeated restriction fragments whose molecular organization is apparently specific to the human X chromosome. This fragment, identified as an ethidium bromide-staining 2.0 kilobase (kb) band in BamHI-digested DNA from a Chinese hamster-human somatic cell hybrid containing a human X chromosome, has been cloned into pBR325 and characterized. The 2.0 kb repeated family has been assigned to the Xp11 leads to Xq12 region on the X by Southern blot analysis of somatic cell hybrids and is predominantly arranged in tandem clusters of up to seven 2.0 kb monomers. Homologous DNA sequences, not organized as 2.0 kb BamHI fragments, are found elsewhere on the X chromosome and on at least some autosomes, but are not found on the Y chromosome. From a dosing experiment using various amounts of the cloned repeat, we estimate that there are 5,000-7,500 copies of the 2.0 kb BamHI repeat per haploid genome. Since the vast majority, if not all, of these are confined to the X chromosome, this repeated DNA family must account for 5-10% of all X chromosome DNA and must constitute the major sequence component of the pericentromeric region of the X.
TL;DR: DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon and revealed the presence of two open reading frames past the last known gene of the spc operon.
Abstract: The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon.
TL;DR: The nucleotide sequence of the coding region and the 5' and 3' flanking regions are determined and potential sites for glycosylation of the secreted invertase are identified.
Abstract: The yeast SUC2 gene is a structural gene for both the secreted and intracellular forms of invertase. We have determined the nucleotide sequence of the coding region and the 5' and 3' flanking regions. The coding regions for the signal peptide-containing precursor to secreted invertase and for the intracellular invertase begin at different initiation codons within the SUC2 gene but share the same reading frame. The amino acid sequences predicted for the two forms of invertase from the nucleotide sequence are consistent with the properties of the purified enzymes. Potential sites for glycosylation of the secreted invertase are identified.
TL;DR: All the detected multiple histone mRNAs are coordinately regulated during the HeLa cell cycle, indicating that post-transcriptional regulation is predominant in late S.
Abstract: Core histone gene expression in HeLa S3 cells has been examined as a function of the cell cycle using cloned human histone gene probes. Total cellular histone mRNAs were analyzed by Northern blot analysis, and their relative abundance shown to be temporally coupled to DNA synthesis rates in S phase. The in vivo incorporation of 3H-uridine into at least fifteen heterologous histone mRNAs (in one hour pulse intervals at various times in the cell cycle), was monitored by hybrid selection. Hybridized RNAs were eluted and resolved electrophoretically to give both a quantitative and qualitative assay for multiple mRNA species. Maximal incorporation of 3H-uridine into histone mRNAs precedes their maximal accumulation, indicating that transcriptional regulation is predominant in early S phase. The turnover of histone mRNAs in late S occurs in the presence of a reduced apparent transcription rate, indicating that post-transcriptional regulation is predominant in late S. All the detected multiple histone mRNAs are coordinately regulated during the HeLa cell cycle.
TL;DR: It is concluded that a single tetA gene mediates resistance in each of these tet determinants of plasmid RP1 and transposon Tn1721.
Abstract: Nucleotide sequences of the homologous tetracycline resistance (tet) determinants of plasmid RP1 and transposon Tn1721 have been determined. Two open reading frames of divergent polarity have been assigned to a regulatory gene (tetR) and a gene encoding a resistance protein (tetA). The intercistronic region contains appropriate regulatory and transcription signals. The tetR gene can code for a protein of 216 amino acids (deduced mol.wt. 23,288) and the tetA gene for a protein of 399 amino acids (deduced mol. wt. 42,205). Based on the deduced amino acid sequence, the tetA proteins of RP1/Tn1721 are 78% homologous with that of pBR322 and 45% homologous with that of Tn10. We conclude that a single tetA gene mediates resistance in each of these tet determinants.
TL;DR: A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described, which results in the production of a wide range of stable phosphoramidates in high yield.
Abstract: A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.
TL;DR: Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo by 'probe' sequences inserted in a very small plasmid vector and introduced into recombination-proficient bacterial cells.
Abstract: Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo. In this method 'probe' sequences are inserted in a very small plasmid vector and introduced into recombination-proficient bacterial cells. Genomic bacteriophage libraries are propagated on the cells, and phage bearing sequences homologous to the probe acquire an integrated copy of the plasmid by reciprocal recombination. Phage bearing integrated plasmids can be purified from the larger pool of phage lacking
plasmid integrates by growth under the appropriate selective conditions.
TL;DR: A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank.
Abstract: A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.
TL;DR: Besides the nicking-closing (topoisomersase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei and activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication.
Abstract: Besides the nicking-closing (topoisomerase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei. The enzyme, partially purified, is able to catenate in vitro closed DNA circles in a magnesium-dependent, ATP-dependent, histone H1-dependent reaction, and to decatenate in vitro kinetoplast DNA networks to yield free minicircles in a magnesium-dependent and ATP-dependent reaction. It is largely similar to other eukaryotic type II topoisomerases in its requirements, and presumably belongs to this class of enzymes. Type I and type II activities were measured in rat liver nuclei as a function of regenerating time after partial hepatectomy: type I activity was not significantly changed during this process. In contrast, type II activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication.
TL;DR: Analysis of the sequences of the genes and identification of at least three different classes of duplication units interspersed throughout the five gene cluster suggests that the cluster evolved quite recently and that the mechanism of gene duplication involved homologous but unequal exchange between middle repetitive elements of the Alu family.
Abstract: The structure of the human growth hormone gene cluster has been determined over a 78 kilobase region of DNA by the study of two overlapping cosmids. There are two growth hormone genes interspersed with three chorionic somatomammotropin genes, all in the same transcriptional orientation. One of the growth hormone genes lies in an active chromatin conformation in the pituitary and at least one of the chorionic somatomammotropin genes lies in an active chromatin conformation in the placenta. The two groups of genes are highly homologous throughout their 5' flanking and coding sequences, but diverge in their 3' flanking regions which raises the paradox of how genes so similar in structural and flanking sequences can be so differentially regulated. Analysis of the sequences of the genes and identification of at least three different classes of duplication units interspersed throughout the five gene cluster suggests that the cluster evolved quite recently and that the mechanism of gene duplication involved homologous but unequal exchange between middle repetitive elements of the Alu family.
TL;DR: The gene coding for amylase has been isolated and sequenced from Bacillus subtilis by cloning in lambda Charon4A and pBR322 and shows a large open reading frame with a translated molecular weight of 72,800 and a presumed signal sequence of approximately thirty-two amino acids.
Abstract: The gene coding for amylase (EC.3.2.1.1) has been isolated and sequenced from Bacillus subtilis by cloning in lambda Charon4A and pBR322. The entire coding sequence and large preceding and following regions, comprising the presumed transcriptional and translational regulatory regions, were sequenced. The coding sequence shows a large open reading frame with a translated molecular weight of 72,800 and a presumed signal sequence of approximately thirty-two amino acids. When the intact gene is present in Escherichia coli, it confers the ability to degrade starch, indicating that the gene is expressed in a functional state.
TL;DR: The results from these studies indicate that sequence selective binding occurs within a small aperture of solution conditions, and the impact that porphyrins' binding has upon the structure of DNA is discussed.
Abstract: The large meso-substituted porphine, meso-tetra(4-N-methylpyridyl)porphine has been identified as a DNA-interactive ligand with a capacity for intercalation (1,2). Subsequently, the 2-N-methyl, 3-N-methyl and N-trimethylanilinium analogues of this porphyrin intercalator have been obtained for physico-chemical analyses (absorption spectroscopy, viscometry, circular dichroism, unwinding of supercoiled DNA). In this paper we discuss the factors affecting the character of porphyrin binding (intercalative, as is the case for the 4-N-methyl and 3-N-methyl porphines, versus non-intercalative, as is the case for the 2-N-methyl and N-trimethylanilinium porphines) and the impact that porphyrins' binding has upon the structure of DNA. The molecular conformation of the porphyrin ligand varies slightly within this series so that the ability of a given porphyrin to intercalate may be correlated with the arrangement of charged groups, the planarity of the porphine ring and the effective width of the individual molecules. The results from these studies indicate that sequence selective binding occurs within a small aperture of solution conditions.
TL;DR: Surprisingly, it was found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.
Abstract: We have constructed deletion and point mutations within the Simian virus 40 (SV40) early promoter region which contains two tandemly repeated 21 bp sequences and a related 22 bp sequence (the "upstream" 21 bp repeat region). After transfection into permissive CV-1 cells and non-permissive mouse 3T3-4E cells, the effect of the mutations on early gene expression was studied by measuring T-antigen production, using indirect immunofluoresence. Our results demonstrate that the 21 bp repeat region, and in particular the six GC-rich motifs 5'-CCGCCC-3' which are repeated in this region constitute an important element of the SV40 early promoter. Surprisingly, we found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.
TL;DR: A new approach is described which will allow the simultaneous synthesis of large numbers of pre-defined oligonucleotide chains using noninterchangeable polymeric entities from each of which enough OD units can be isolated after completion of the syntheses.
Abstract: A new approach is described which will allow the simultaneous synthesis of large numbers of pre-defined oligonucleotide chains. No machine aid is needed. The simultaneous syntheses can be performed by one person and do not require much more time than is currently needed for the synthesis of just one oligonucleotide in existing strategies. The general idea is the following: One uses noninterchangeable polymeric entities from each of which enough OD units can be isolated after completion of the syntheses. Whenever growing chains on different entities have to be elongated with the same building block these entities are gathered in the same reaction vessel. After such a common reaction cycle the entities are separated and now combined according to the next common building blocks etc. The practicability of this approach is demonstrated by the synthesis of d(T-A-A-T-A-T-T-A) and d(T-A-G-T-A-C-T-A) on cellulose filter disks following the phosphotriester approach.