Showing papers in "Nucleic Acids Research in 1988"
TL;DR: A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure that yielded quantities comparable to those obtained from phenol-chloroform extractions, rendering the entire process of RFLP analysis free of toxic materials.
Abstract: One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deprotein-izing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl t 400 mM NaCl and 2 mM Na 2 EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10Z SDS and 0.5 ml of a protease K solution (1 mg protease K in 1Z SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pi TE buffer (10 mM Tris-HCl, 0.2 mM Na 2 EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our non-isotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.
19,905 citations
TL;DR: An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate and easy to use on microcomputers, based on the conventional dynamic-programming method of pairwise alignment.
Abstract: An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment. Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is illustrated by an example: a global alignment of 39 sequences of cytochrome c.
5,208 citations
TL;DR: E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation), and most of the surviving cells are competent with up to 80% transformed at high DNA concentration.
Abstract: E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.
2,868 citations
TL;DR: These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA.
Abstract: Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.
2,471 citations
TL;DR: This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus and it is demonstrated the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.
Abstract: The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.
1,323 citations
TL;DR: A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed in this article, which eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation.
Abstract: A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.
1,321 citations
1,035 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
887 citations
TL;DR: Results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.
Abstract: DNA polymerases catalyze the addition of deoxyribonucleotides onto DNA primers in a template-directed manner. The requirement for template instruction distinguishes these enzymes from other nucleotidyl transferases, such as terminal deoxynucleotidyl transferase, that do not utilize a template. An oligonucleotide substrate was used to characterize a novel, non-templated nucleotide addition reaction carried out by DNA polymerases from a variety of procaryotic and eucaryotic sources. The products of the reaction, in which a deoxyribonucleotide was added to the 3' hydroxyl terminus of a blunt-ended DNA substrate, were analyzed by electrophoresis on high resolution, denaturing polyacrylamide gels. DNA polymerase from Thermus aquaticus, polymerase alpha from chick embryo, rat polymerase beta, reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae all carried out the blunt-end addition reaction. The reaction required a duplex DNA substrate but did not require coding information from the template strand. These results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.
877 citations
TL;DR: The syntheses, melting temperatures, and nuclease susceptibilities of a series of phosphorothioate ODN analogs are reported, providing a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.
Abstract: We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.
TL;DR: DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene and found a mutation in codon 12 of the KRAS gene, confirming the findings of Almoguera et al.
Abstract: DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene. Formalin-fixed and paraffin-embedded tissue was used directly in an in vitro amplification reaction to expand the relevant RAS sequences. The mutations were detected by selective hybridization using mutation-specific synthetic oligonucleotides. In 28 of the 30 patients we found a mutation in codon 12 of the KRAS gene. This result confirms the findings of Almoguera et al. [Cell 53 (1988) 549-554] that KRAS mutations occur frequently in adenocarcinomas of the exocrine pancreas. The mutations are predominantly G-T transversions, in contrast to the KRAS mutations in colon tumors which are mainly G-A transitions. Furthermore, in a portion of the tumors the mutation appears to be homozygous.
TL;DR: The pSG5 vector as mentioned in this paper was constructed by combining the eukaryotic expression vector, pKCR2, and the high copy plasmid vector Bluescribe Ml3+ (Stratagene).
Abstract: The ability to engineer specific modifications within the coding region of a structural gene is a powerful tool with which to study the relationship between protein structure and function. The process of site-directed mutagenesis involves a) mutation of the target gene, b) verification of the mutation by sequencing and c) expression of the mutated gene. We describe here a vector, pSG5 (Fig.lA), in which each of these steps may be performed. pSG5 was constructed essentially by combining the eukaryotic expression vector, pKCR2 (1), and the high copy plasmid vector Bluescribe Ml3+ (Stratagene). The principle features of pSG5 are a) unique EcoRI and BamHI restriction enzyme sites for insertion of cDNAs, b) production of single stranded DNA containing the antisense strand of the structural gene for mutagenesis and sequencing, c) high yields of double stranded DNA, d) in vivo expression from the SV40 early promoter and e) in vitro expression from the T7 promoter situated just upstream of the cDNA insertion site. Expression from pSG5 was tested after insertion of a cDNA encoding the human oestrogen receptor (2) (ER) into the EcoRI site to produce pSG5-ER. Fig.IB shows an SDS polyacrylamide gel of the 66Kd ER protein synthesised in a rabbit reticulocyte cell-free translation cocktail programmed with mRNA transcribed from the T7 promoter of either Bluescribe-ER (BSM-ER (3)) or pSG5-ER. The ER when expressed in HeLa cells stimulates transcription from oestrogen-responsive 'reporter' genes (4) (vitellogenin-TK-globin (VTG) in Fig. 1C). Using quantitative SI nuclease analysis with an internal reference gene (RXF in Fig.lC), the ER expressed from either pSG5-ER or pKCR2-ER stimulates gene transcription equally well indicating that equivalent levels of ER are expressed in vivo using either of these two vectors.
TL;DR: These trends for codon usage are illustrated for six species whereCodon usage has been examined in detail, by presenting the pooled codon used for the 10% of genes at either end of the major trend.
Abstract: The genetic code is degenerate, but alternative synonymous codons are generally not used with equal frequency. Since the pioneering work of Grantham's group it has been apparent that genes from one species often share similarities in codon frequency; under the "genome hypothesis" there is a species-specific pattern to codon usage. However, it has become clear that in most species there are also considerable differences among genes. Multivariate analyses have revealed that in each species so far examined there is a single major trend in codon usage among genes, usually from highly biased to more nearly even usage of synonymous codons. Thus, to represent the codon usage pattern of an organism it is not sufficient to sum over all genes as this conceals the underlying heterogeneity. Rather, it is necessary to describe the trend among genes seen in that species. We illustrate these trends for six species where codon usage has been examined in detail, by presenting the pooled codon usage for the 10% of genes at either end of the major trend. Closely-related organisms have similar patterns of codon usage, and so the six species in Table 1 are representative of wider groups. For example, with respect to codon usage, Salmonella typhimurium closely resembles E. coli, while all mammalian species so far examined (principally mouse, rat and cow) largely resemble humans.
TL;DR: An empirical relation between the degree of bending and the altered electrophoretic mobility in polyacrylamide gels that allows estimation of protein-induced bends is generated.
Abstract: Protein-induced DNA bending is an important element in the structure of many protein-DNA complexes, including those involved in replication, transcription, and recombination. To understand these structures, the path followed by the DNA in each complex must be established. We have generated an empirical relation between the degree of bending and the altered electrophoretic mobility in polyacrylamide gels that allows estimation of protein-induced bends. This technique has been used to analyze 17 different protein-DNA complexes formed by six proteins including the four proteins involved in lambda site-specific recombination. The simplicity of this technique should make it useful in estimating angles for the construction of models of protein-DNA complexes and readily applicable to many systems where questions of higher-order structure are important for understanding function.
TL;DR: In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.
Abstract: The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.
TL;DR: The results support the idea that, generally speaking, introns were present in primitive genomes, though in some cases they may have been inserted into pre-existing genes.
Abstract: The lengths of introns and exons in various parts of genes of vertebrates, insects, plants and fungi are tabulated. Differences between the various groups of organisms are apparent. The results are discussed and support the idea that, generally speaking, introns were present in primitive genomes, though in some cases they may have been inserted into pre-existing genes.
TL;DR: Hypervariable minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA.
Abstract: Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.
TL;DR: The sequences show that this ancient individual belonged to a mitochondrial lineage that is rare in the Old World and not previously known to exist among Native Americans, bringing to three the number of maternal lineages known to have been involved in the prehistoric colonization of the New World.
Abstract: Pieces of mitochondrial DNA from a 7000-year-old human brain were amplified by the polymerase chain reaction and sequenced. Albumin and high concentrations of polymerase were required to overcome a factor in the brain extract that inhibits amplification. For this and other sources of ancient DNA, we find an extreme inverse dependence of the amplification efficiency on the length of the sequence to be amplified. This property of ancient DNA distinguishes it from modern DNA and thus provides a new criterion of authenticity for use in research on ancient DNA. The brain is from an individual recently excavated from Little Salt Spring in southwestern Florida and the anthropologically informative sequences it yielded are the first obtained from archaeologically retrieved remains. The sequences show that this ancient individual belonged to a mitochondrial lineage that is rare in the Old World and not previously known to exist among Native Americans. Our finding brings to three the number of maternal lineages known to have been involved in the prehistoric colonization of the New World.
TL;DR: Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels, and a chimeric gene containing the metallothsionein promoter shows a similar induction when transformed into the cells.
Abstract: The promoter from the metallothionein gene may be a useful conditional promoter for the construction of chimeric genes to be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed chimeric gene containing the metallothionein promoter were examined. Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels. The level of induction depends on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing the metallothionein promoter shows a similar induction when transformed into the cells.
TL;DR: This work has defined the 13 bp palindrome GGTCACAGTGACC as a minimal functional estrogen responsive element (ERE), which binds estrogen receptor preferentially in vitro and point mutations within the ERE decrease its affinity for the estrogen receptor and result in a complete loss of estrogen inducibility.
Abstract: Sequences located upstream of the transcription initiation site of the Xenopus vitellogenin A2 (vit A2) gene contain a hormone dependent enhancer that confers estrogen control to the heterologous thymidine kinase (tk) promoter. As a minimal functional estrogen responsive element (ERE), we have defined the 13 bp palindrome GGTCACAGTGACC. This ERE binds estrogen receptor preferentially in vitro. Although the ERE shares some structural features with the glucocorticoid responsive element (GRE) it is distinct from this element since it neither binds glucocorticoid receptor in vitro nor does it confer glucocorticoid inducibility to a fusion gene. Point mutations within the ERE decrease its affinity for the estrogen receptor and result in a complete loss of estrogen inducibility.
TL;DR: It appears that the precursor was also digested with Haell which generated the Haell Cm resistance segment and the Hael fragments found between the Tet and Cm genes.
Abstract: pACYC184 ii a commonly used multicopy cloning vector which was constructed by ligating restriction fragments from pSClOl, Tn9, and pl5A each of which have been previously sequence*! (1,23,4,5). Despite its wide use, the complete nucleotide sequence of pACYC 184 has never been repented. The sequence was completed by using oligonudeotide primers designed to span the junctions between each of the previously sequenced regions. pACYC184is 4244 bp in length with nucleotide 1 corresponding to the EcoRI site in the original map (1). The chtorampbenicol resistance (Cm) segment from Tn9 extends from the Haell site at base 3505 to the Haell site at base 585 with bases 219 (ATG) to 3804 encoding the Cm gene. Part of an IS1 (5) from Tn9 extends from bases 443 to 583. Bases 1494 to 3275 are derived from pSClOl with the tetracycline (Tet) resistance gene encoded by bases 1580 (ATG) to 2770. The pl5A origin of replication extends from bases 581 to 1492. Three fragments; an Alul (3276) to Haell (3368), a Haen (3368) to Haell (3422), and a Haell (3422) to Haell (3505) are located between Cm and Tet gene and are all derived from different regions of the Tet gene. During the construction of pACYCl84 a precursor plasmid, pACYC175, was digested with Haem, Alul, and Hindi, to remove extraneous DNA and to reduce the size of the plasmid. It appears that the precursor was also digested with Haell which generated the Haell Cm resistance segment and the Haell fragments found between the Tet and Cm genes. The underlined sequence was determined while the rest of the sequence was taken from the published sequences of pSClOl (2), Tn9 (3,4), and pl5A (5).
TL;DR: A 15-residue sequence motif has been found in many polymerases from various species and involving DNA and RNA dependence and product and may be important in polymerase function by acting directly in catalysis and/or by binding magnesium.
Abstract: A 15-residue sequence motif has been found in many polymerases from various species and involving DNA and RNA dependence and product. The motif is characterized by a Tyr-Gly-Asp-(Thr)-Asp core flanked by hydrophobic spans five residues in length. An mRNA maturase segment is also suggested to display the motif pattern. The aspartates may be important in polymerase function by acting directly in catalysis and/or by binding magnesium.
TL;DR: Mutational analysis of the left half of the palindrome showed that a perfect dyad symmetry is not required for optimum activity as a steroid response element, and the minimum sequence requirements for a hormone response were analysed.
Abstract: We have characterized steroid response elements in mouse mammary tumour virus (MMTV) by transient transfection. Four partial inverted repeats of the sequence TGTTCT function as response elements for androgen, as well as for glucocorticoid and progestins, although the relative hormone inductions mediated by each oligonucleotide were different. Mutational analysis of the left half of the palindrome showed that a perfect dyad symmetry is not required for optimum activity as a steroid response element. To investigate potential interactions between steroid receptors and transcription factors we have analysed the minimum sequence requirements for a hormone response. Interestingly, a single 15 bp steroid response element and a TATA box are sufficient for steroid inductions. When the distance between the two elements was increased by up to two turns of the helix the hormone induction initially increased and then gradually declined with no obvious periodicity.
TL;DR: This technique provides a convenient and reproducible method for the preparation and storage of competent bacterial cells that yields high transformation efficiencies but is simpler and more convenient than other techniques.
Abstract: Several effective methods for bacterial cell transformation have previously been described. These methods involve treatment of cells with CaCl2 (1-5) or cobalt hexamine chloride (6). Agents such as polyethylene glycol (PEG) have also been shown to induce bacterial cell transformation (7). We now describe a new method for the preparation of competent bacterial cells that yields high transformation efficiencies but is simpler and more convenient than other techniques. Bacterial cells are grown to the early log phase (ODgrjo = 0.3-0.6) in LB broth, then pelleted by centrifugation (1,000 X g for 10 mins at 4°C). The cells are then resuspended in l/10th volume of transformation and storage buffer (TSB): LB broth (pH 6.1) containing 10% PEG (MW-3,350), 5« DMSO and 20 mH Mg + + (lOmM MgCl2 + 10 mH MgSO4) at 4°C, and incubated on ice for approximately 10 mins. For transformation, 0.1 ml aliquots of the cells are pipetted into cold polypropylene tubes and mixed with 100 pg of plasmid DNA. The cells are returned to ice for 5-30 mins. Heat shock is not necessary. Next, cells are grown to permit expression of the antibiotic resistance gene [0.9 ml of TSB with 20 mM glucose is added, and the cells incubated at 37°C with shaking (225 rpm) for 60 mins] and plated on antibiotic-containing agar plates for selection of transformants. Using this protocol, we obtain transformation efficiencies of up to 2 X 10 transformants per microgram of DNA. Moreover, cells in TSB can be frozen (in a dry ice/ethanol bath) and stored at -70°C for use at a later date without a significant loss of cell viability or transformation efficiency. This technique provides a convenient and reproducible method for the preparation and storage of competent bacterial cells.
TL;DR: Evidence is reviewed suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.
Abstract: The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.
TL;DR: The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution.
Abstract: The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.
TL;DR: The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
Abstract: N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
TL;DR: The introduction of a boundary constraint results in an array of regularly spaced nucleosomes at nonrandom locations, similar to the arrays reported for some genes and other chromosomal regions.
Abstract: Expressions are derived for distributions of nucleosomes in chromatin. Nucleosomes are placed on DNA at the densities found in bulk chromatin, and their locations are allowed to vary at random. No further assumptions are required to simulate the periodic patterns of digestion obtained with various nucleases. The introduction of a boundary constraint, due for example to sequence-specific protein binding, results in an array of regularly spaced nucleosomes at nonrandom locations, similar to the arrays reported for some genes and other chromosomal regions.