Long-range mapping of gaps and telomeres with RecA-assisted restriction endonuclease (RARE) cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is a method to perform sequence-specific cleavage of genomic DNA, and is useful in physical mapping studies. After making two modifications, we have applied this method to mapping large regions of DNA in several cell types, including a notorious gap near the Huntington disease (HD) locus on chromosome 4. RARE cleavage fragments were analysed by pulsed field gel electrophoresis and Southern blotting and the distances between cleavage sites determined with accuracy. Using RARE cleavage, the gap measured was less than 60 kilobases in length. RARE cleavage is also a straightforward technique to map the distance from a marker to a telomere. The terminal 1.7 megabases of several HD and control cell lines were mapped with no large differences between cell lines in this region.

Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage.

Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.

Survey of genetic alterations in gastrinomas.

Gastrinomas are rare gastrin-secreting endocrine tumors that usually arise in the duodenum or pancreas and, if untreated, can cause severe peptic ulcers or metastatic disease. Although most tumors are sporadic they are especially common in patients with multiple endocrine neoplasia type 1 (MEN1), and most studies of these tumors have focused on the role of the MEN1 gene. Although the gene is commonly altered in sporadic tumors, this finding is not universal, and it is highly likely that other genetic defects play a significant role. In the present study, an in-depth analysis of the DNA of eight tumors was carried out in an effort to localize these areas. The experiments consisted of an analysis of 400 microsatellite marker loci distributed evenly throughout the human genome, and the results were confirmed with comparative genomic hybridization. Whereas deletions encompassing the MEN1 gene were seen in two tumors, the most striking result was multiple large rearrangements on chromosome 1 in two of the tumors with hepatic metastases. In several instances, an individual tumor had abnormalities of every informative maker on a given chromosome, presumably as a result of aneuploidy affecting that chromosome. Such defects were only seen in the four large or aggressive tumors, and the total number of chromosomes affected in a tumor ranged from 1 to a high of 13 in a patient who had an unusually aggressive tumor This tumor also showed microsatellite instability, and this is the first report of such a defect in gastrinomas. This study implicates chromosome 1 defects, aneuploidy, and perhaps mismatch repair defects as importan features of gastrinomas; deletions involving the MEN1 gene were con firmed, but the rest of the genome was free of large deletions or amplifications.

Flexible genetic engineering using RecA protein.

With the explosion in genetic information and almost complete sequencing of the human genome, a shift in the experimental goals of molecular biologists is occurring. Instead of focusing on single genes, current attempts seek to divine the interactions of several genes and sequences. This requires increasingly complex genetic constructs and manipulations, often of very large DNA constructs, and these can be made with RecA protein-based techniques. When RecA protein combined with an oligonucleotide acts as a sequence-specific "masking tape" to block DNA from the action of DNA modifying enzymes, and can be used to direct the cleavage of DNA at single predetermined restriction endonuclease sites. This reaction is called RecA-Assisted Restriction Endonuclease (RARE) Cleavage. The reverse reaction, known as RecA-Assisted Ligation, can be used to join any two desired fragments. When one of those fragments is a vector, a desired fragment can be cloned directly without constructing a genomic library. The reagents and equipment needed are relatively inexpensive, and almost any desired genetic construct up to about 300 kb in size can be made in a straightforward manner.

Nuclear Overhauser effect studies of the conformations and binding site environments of deoxynucleoside triphosphate substrates bound to DNA polymerase I and its large fragment.

The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR studies of conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I.

The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)

Metal content of DNA polymerase I purified from overproducing and wild type Escherichia coli.

DNA polymerase I purified from both E. coli strain B, and from an overproducing E. coli stain lysogenized with a lambda pol A phage were analyzed for metal content. After gel filtration to remove loosely bound metals, DNA polymerase I from both strains contained less than or equal to 0.2 gm atoms Zn2+/mole enzyme and 0.09 to 0.7 Mg2+/mole enzyme. Substoichiometric amounts of Fe, Co, Ni (less than or equal to 0.2 gm atoms), and Mn (less than or equal to 0.1 gm atoms) were detected. Since the metal content does not correlate with enzymatic activity, we conclude that DNA polymerase I is not a metalloenzyme.

Sequence-specific ligation of DNA using RecA protein.

A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.

High-density allelotype of the commonly studied gastric cancer cell lines.

Gastric cancer is one of the leading causes of death from cancer throughout the world, and studies to elucidate the genetic defects found in this type of cancer are growing in number. Increasingly sophisticated techniques and the sequencing of the human genome have had an impact on the scope of such studies. While the use of tumor specimens remains popular, more emphasis is being placed on cell lines as model systems where specific data can be directly combined with results from other studies. This article describes a genetic survey of the most widely used gastric adenocarcinoma cell lines. The allelotype at 351 polymorphic loci in 14 cell lines was obtained, and the results from the 4,900 polymerase chain reactions are displayed. In addition to confirming loss of heterozygosity on chromosome arms 6p, 7q, 17p, and 18, additional deletions on arm 5p and the pericentromeric regions of chromosomes 1 and 10 were detected. Areas that might contain homozygous deletions or amplifications also were mapped. The rate of microsatellite instability was quantified and shown to vary greatly among the different cell lines. Most important, this study serves as a genetic scaffold for the integration of past and future studies on the nature of the genetic defects in gastric cancer.

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment.

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)