The use of exonuclease III for preparing single stranded DNA for use as a template in the chain terminator sequencing method

Mouse cells deficient in the enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) have been transfected with total human DNA, and cells producing human enzyme were isolated by growth in selective medium. DNA from several such cell lines has been used to generate secondary transfectants that make human HPRT. Blots of the DNA of these secondary cells have been hybridized with total human DNA probes or with cloned human Alu sequences, and one of several common bands has been cloned in pBR322. Colonies of transformed Eacherichia coli containing human sequences were detected by their homology with human DNA, and subclones of resulting recombinant plasmids were prepared. Two subclones free of Alu sequences were found to contain human sequences that hybridized to human X chromosome DNA. One of these, pBRI.5, also hybridized to a single RNA band on gel blots of human and secondary transfectant cytoplasmic poly(A)+RNA but not to RNA from the parent mouse cell line. These results indicate that these clones represent human HPRT HPRT Assays. Cell lines were assayed for the presence of mouse or human HPRT on isoelectric focusing gels as described (9). DNA Preparation. Human placental DNA was prepared by the method of Graham (10). DNA from tissue culture cells was prepared as described by Pellicer et aL (11). Plasmid DNA was prepared from Escherichia coli by conventional methods (12).RNA Preparation. Cytoplasmic RNA was prepared according to Levis and Penman (13), and poly(A)+RNA was selected by oligo(dT)-cellulose chromatography (14).Blots. DNA gels were blotted according to Southern (15); acid pretreatment (16) was used when transfers included high molecular weight (25 kb) bands. RNA was electrophoresed in agarose gels and blotted to nitrocellulose filters according to Thomas (17). High-stringency hybridization conditions were: 0.5 M NaCl, 20 mM Hepes at pH 7.4, 0.1 mM EDTA, 30% (vol/vol) 5038The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

show abstract

“…In some cases, human DNA was labeled by Exo III treatment followed by fill-in synthesis with reverse transcriptase (27).…”

mentioning

confidence: 99%

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Jolly¹,

Esty²,

Bernard³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Similarly, the digestion pattern of the 2.8-kb hCS fragment (Fig. 3B) (15). An autoradiograph of a sequencing gel is shown in Fig.…”

Section: Methodsmentioning

confidence: 81%

Structure of genes for human growth hormone and chorionic somatomammotropin.

Fiddes¹,

Seeburg²,

Denoto³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A 2.6-kilobase (kb) EcoRI restriction endonuclease fragment containing human growth hormone (hGH; somatotropin) gene sequences and a 2.8-kb EcoRI fragment containing human chorionic somatomammotropin (hCS; choriomammotropin) gene sequences have been identified by hybridization to cloned cDNA. Human DNA was cleaved with EcoRI and fractionated by preparative agarose gel electrophoresis; DNA in the size range 2-3 kb was ligated to Xgt WES'XB DNA and viable recombinant bacteriophage were recovered by in vitro packaging. After infection of Escherichia coli and screening of phage plaques, single isolates of hGH and hCS gene sequences were obtained. Restriction endonuclease mapping showed that the hGH gene contains three intervening sequences interrupting the coding sequence. Partial DNA sequence analysis of the hGH gene, obtained by the chain termination method, confirmed the location of the intervening sequences and the identity of the fragment. Human growth hormone (hGH) and human chorionic somatomammotropin (hCS) are two closely related polypeptide hormones which have more than 80% of their 191 amino acids in common (1, 2). They have different biological activities and are synthesized in different tissues: hGH in the pituitary and hCS in the placenta. The genes coding for these two hormones provide a good system for studying the organization of structurally related sequences and their tissue-specific expression.A 550-base-pair Hae III fragment cDNA clone coding for amino acids 24-191 of the hCS sequence has been described (3). Recently, the analogous 550-base-pair Hae III fragment coding for hGH has also been cloned and its nucleotide sequence has been determined (unpublished data). The nucleotide sequences of these two fragments are 93% homologous.Further analysis of this system requires the isolation of genomic DNA fragments that contain the entire coding sequences and the regulating elements that may be involved in the differential expression of the genes. This report describes the construction of genomic DNA clones for sequences of both the hGH and hCS genes by utilizing bacteriophage Xgt WES-XB as the vector (4) and the bacteriophage X in vitro packaging system of Blattner et al. (5). MATERIALS AND METHODSHuman Placental DNA. High molecular weight DNA was extracted from human placenta obtained by caesarian section. The frozen tissue was dispersed in a blender, treated with proteinase K, extracted with phenol/chloroform, and then incubated sequentially with RNase A and proteinase K (6). After extraction with phenol/chloroform, the DNA was precipitated with ethanol.Preparative Agarose Gel Electrophoresis. EcoRI-digested human placental DNA (17.5 mg) was fractionated on a continuous elution, horizontal, 0.8% agarose gel, essentially as described by Polsky et al. (6) except that Seakem HGT (P) agarose was used because this appeared to contain fewer contaminants that inhibit subsequent enzymatic steps. The DNA was collected in approximately 80 fractions, and aliquots of these were assayed by analytical aga...

show abstract

“…Supercoiled plasmid DNA (5 ,g) was digested with BamH to completion in a total volume of 20 Ml. Then, 3 Ml of 10-fold concentrated exonuclease buffer (10 mM MgCIJ100 mM dithiothreitol/700 mM Tris, pH 7.5) was added followed by 5 units of exonuclease III, and the mixture was digested in sealed capillaries for 204 hr at 37°C (14). The digestions were terminated by heating to 68°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

The nucleotide sequence surrounding the replication terminus of R6K.

Bastia¹,

Germino²,

Crosa³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The replication terminus of the plasmid R6K has been cloned into the single-stranded DNA phage vector M13mpS and also into the plasmid vectors pBR313 and pBR322. By using single-stranded DNA templates prepared from the recombinant DNA clones, the sequence of 215 base pairs of DNA containing the replication terminus has been determined. The DNA sequence of the region ofthe terminus does not contain any 2-fold rotational symmetry. Therefore, folding of the DNA at the region of the terminus is unlikely to be a cause -for replication termination. Interaction of a host-specified protein(s) with the sequence of the replication terminus is probably the basis of the mechanism of replication termination.In spite of extensive work, the molecular mechanism of termination of DNA replication still remains unclear. In efforts to elucidate the mechanism of termination of replication, analysis of the DNA sequences involved in replication termination and identification of proteins that interact with the replication terminus are of utmost importance.The drug-resistance factor R6K which carries genes for ampicillin and streptomycin resistance (1) is a model system for this study because its replication is asymmetrically bidirectional. The asymmetry in the bidirectional replication is manifested in the confluence of the two moving forks of the replicating chromosome at a point that is not located at 1800 from the replication origins (2, 3). The asymmetrically bidirectional replication strongly suggested the existence of a genetically determined terminus of replication.In this paper we report the cloning ofthe replication terminus into the vector M13mp5, its precise localization in a 215-basepair DNA fragment, and analysis of its nucleotide sequence. The nucleotide sequence of the replication terminus suggests a mechanism of termination that probably involves interaction of the terminus sequence of the plasmid with one or more proteins specified by the host genome. MATERIAL AND METHODSBacterial, Plasmid, and phage Strains. The bacterial strain JC411 thy-was obtained from D. Helinski and the strain 71-18, from J. Messing (4). The plasmid vectors pBR313 and pBR322 (5) were obtained from H. Boyer through R. Curtiss III. The single-stranded DNA phage vector M13mp5, which contains a single HindIII site was a gift from J. Messing.Chemicals, Enzymes, and Labeled Precursors. dNTPs (Sigma), dideoxynucleotide triphosphates (P-L Biochemicals), [a-32P]dATP (100-300 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels; ICN), 5-bromo-4-chloroindolyl /3-galactoside (Sigma) and isopropyl 13-thiogalactoside; (Sigma) were obtained as indicated.The restriction enzymes HindIII (6), Hpa II (7), Alu 1 (8), Hinf (9), Hae III and Hae 11 (10), and Hha (11) were purified according to published procedures. DNA polymerase I holoenzyme and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim. Exonuclease III and BamHI were purchased from New England BioLabs. T4 ligase was purified as described by Weiss (12).Molecular Cloning. These experime...

show abstract

The use of exonuclease III for preparing single stranded DNA for use as a template in the chain terminator sequencing method

Cited by 98 publications

References 9 publications

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Isolation of a genomic clone partially encoding human hypoxanthine phosphoribosyltransferase.

Structure of genes for human growth hormone and chorionic somatomammotropin.

The nucleotide sequence surrounding the replication terminus of R6K.

Contact Info

Product

Resources

About