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Showing papers on "Lambda phage published in 1990"


Journal ArticleDOI
30 Aug 1990-Nature
TL;DR: The results show that the λ prophage is more tran-scriptionally active than has long been assumed, and suggest that lysogeny may generally have a role in bacterial survival in animal hosts, and perhaps in pathogenesis.
Abstract: ALTHOUGH phage λ represents a well studied biological system, it has certain features that remain obscure. Among these is the function of the roughly one third of the phage genome dispensable for growth in the laboratory, yet retained despite undoubted pressure to economize1. Here we report that these 'accessory' sequences contain two genes which are expressed during lysogeny, and encode host-cell envelope proteins. One of these is lom (ref. 2), the product of which is found in the bacterial outer membrane, and is homologous to virulence proteins of two other enterobacterial genera. The other gene, previously unidentified, we designate bor. Expression of bor significantly increases the survival of the Escherischia coli host cell in animal serum. This property is a well known bacterial virulence determinant3,4—indeed, bor and its adjacent sequences are highly homologous to the iss serum resistance locus of the plasmid ColV2-K94, which confers virulence in animals5,6. These results show that the λ prophage is more tran-scriptionally active than has long been assumed, and suggest that lysogeny may generally have a role in bacterial survival in animal hosts, and perhaps in pathogenesis.

181 citations


Journal ArticleDOI
01 Mar 1990-Gene
TL;DR: Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described.

140 citations


Journal ArticleDOI
TL;DR: There are new insights into the way this structure directs critical events during recombination of bacteriophage lambda into the chromosome of E. coli.

124 citations


Journal ArticleDOI
30 Mar 1990-Gene
TL;DR: Two classes of cDNA cloning vectors are described, designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system and the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures.

123 citations


Journal ArticleDOI
TL;DR: A superfamily of proteins encoded by bacterial, phage and eukaryotic genomes and performing a wide range of NTP-dependent functions was delineated by amino acid sequence comparison.

116 citations


Journal ArticleDOI
TL;DR: Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial Rescue efficiencies, permitting quantitative mutational analysis.
Abstract: Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.

115 citations


Journal ArticleDOI
TL;DR: The overview model for packaging and cutting of X DNA which is currently accepted is a random selection of a cos site from among those present on a concatemer for packaging initiation, polarity of chromosome entry into the prohead from the Nul end to the R end, and dependence of cos cutting on the packaging of a minimal length ofDNA.
Abstract: The mature chromosome of bacteriophage X is a linear duplex of a unique sequence 48,502 nucleotide pairs long (34). The 5'-terminal ends of this molecule protrude as self-complementary single-stranded chains of 12 nucleotides which can anneal to generate circles. When a X DNA molecule is injected into the host, it is circularized and closed covalently by DNA ligase action. Early during lytic infection, X DNA undergoes several rounds of replication in which circles generate circles. At later times of infection, rolling-circle replication takes over and individual rings give rise to linear concatemers (3, 8, 9, 28) which may be 10 chromosomal units long (in the absence of packaging). During DNA packaging, monomeric chromosomal units are cut from these replicative oligomers by the enzyme X DNA terminase, which regenerates the unique single-stranded ends by introducing two specific nicks staggered 12 nucleotides apart on opposite strands of the duplex, followed by melting of the joint. The DNA site of action of terminase is called cos. By its interaction with cos, terminase not only catalyzes the formation of ends but, in coupling proheads to DNA, establishes the origin and direction of packaging. The overview model for packaging and cutting of X DNA which is currently accepted is as follows: (i) random selection of a cos site from among those present on a concatemer for packaging initiation, (ii) polarity of chromosome entry into the prohead from the Nul end to the R end, (iii) dependence of cos cutting on the packaging of a minimal length ofDNA, and (iv) processive packaging oftwo or three chromosomes in series from a concatemer (8, 9, 28). For previous reviews on this subject, see references 8, 9, and 28. With a few exceptions, references included in these reviews will not be cited specifically; instead, the reader will be directed to one or more of these three reviews, as the case may be.

113 citations


Journal ArticleDOI
S M Sell1, C Eisen1, Debbie Ang1, Maciej Zylicz1, Costa Georgopoulos1 
TL;DR: Evidence is provided that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C, however, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.
Abstract: Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.

101 citations


Journal ArticleDOI
TL;DR: A description of a lysogen whose prophage is mutated in certain genes, so that the switch is functionally isolated from the rest of the phage genome, and a new method for the measurement of intracellular concentrations is suggested.

96 citations


Journal ArticleDOI
TL;DR: In this article, the T4-type phages of Escherichia coli (T4, T2, TuIa, TIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers.

88 citations


Journal ArticleDOI
TL;DR: The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.

Journal ArticleDOI
TL;DR: The results are compatible with previous proposals that lambda homologous recombination involves the following early steps: generation of double-strand ends by the packaging machinery or by the replication machinery; production of a single-stranded tail with a 3'-hydroxyl end by 5'----3' degradation by lambda exonuclease (red alpha gene product).
Abstract: We have obtained evidence for the repair of double-strand gaps promoted by the Red function of bacteriophage lambda. A double-strand gap was made in one of the two regions of homology in an inverted orientation on a plasmid DNA molecule. The gapped plasmid was introduced into Escherichia coli cells expressing the red alpha (exo) and red beta (bet) genes of lambda. The gap was repaired by DNA synthesis copying an intact duplex. This gap repair was sometimes accompanied by reciprocal recombination (crossing over). The gap stimulated recombination about 100-fold. Our results are compatible with previous proposals that lambda homologous recombination involves the following early steps: (i) generation of double-stranded ends by the packaging machinery or by the replication machinery; (ii) production of a single-stranded tail with a 3'-hydroxyl end by 5'----3' degradation by lambda exonuclease (red alpha gene product); (iii) pairing of the single-stranded tail with a complementary strand from a homologous duplex with the help of beta protein (red beta gene product); (iv) priming of DNA synthesis at this 3'-hydroxyl end to copy the second DNA molecule.

Journal ArticleDOI
TL;DR: Escherichia coli integration host factor (IHF) is a small dimeric protein that binds to a specific DNA consensus sequence and produces DNA bending and enhances the formation of RNA polymerase-promoter closed complexes.

Journal ArticleDOI
01 Nov 1990-Genetics
TL;DR: Two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the Rec BCD enzyme to effect recombination.
Abstract: When one of two infecting lambda phage types in a replication-blocked cross is chi + and DNA packaging is divorced from the RecBCD-chi interaction, complementary chi-stimulated recombinants are recovered equally in mass lysates only if the chi + parent is in excess in the infecting parental mixture. Otherwise, the chi 0 recombinant is recovered in excess. This observation implies that, along with the chi 0 chromosome, two chi + parent chromosomes are involved in the formation of each chi + recombinant. The trimolecular nature of chi +-stimulated recombination is manifest in recombination between lambda and a plasmid. When lambda recombines with a plasmid via the RecBCD pathway, the resulting chromosome has an enhanced probability of undergoing lambda x lambda recombination in the interval into which the plasmid was incorporated. These two observations support a model in which DNA is degraded by Exo V from cos, the sequence that determines the end of packaged lambda DNA and acts as point of entry for RecBCD enzyme, to chi, the DNA sequence that stimulates the RecBCD enzyme to effect recombination. The model supposes that chi acts by ejecting the RecD subunit from the RecBCD enzyme with two consequences. (1) ExoV activity is blocked leaving a highly recombinagenic, frayed duplex end near chi, and (2) as the enzyme stripped of the RecD subunit travels beyond chi it is competent to catalyze reciprocal recombination.

Journal ArticleDOI
TL;DR: Western blot analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody demonstrated that the S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle.
Abstract: Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.

Journal ArticleDOI
TL;DR: All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.
Abstract: We used lambda and plasmid vectors containing the am+ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am+ transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a lambda vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only lambda long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the lambda vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.

Journal ArticleDOI
TL;DR: The results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that thednaJ protein specifically interact with the dnB helicase, which unwinds the duplex DNA template at the replication fork.

Journal ArticleDOI
TL;DR: The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn isdependent on the gene products of comA and comB.
Abstract: Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B subtilis chromosomal DNA was constructed Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB

Journal ArticleDOI
TL;DR: It is suggested that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA,nusB or nusE, but that the complex formed with mutant components differs functionally from wild- type.

Journal ArticleDOI
TL;DR: It is found that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner, which suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.
Abstract: The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B9. The regions corresponding to the C9 core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int. Images

Journal ArticleDOI
TL;DR: During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth.
Abstract: During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.

Journal ArticleDOI
TL;DR: The ratio of effector to inhibitor was much higher in P22 than in lambda and it is proposed that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.
Abstract: Gene 13 of bacteriophage P22 is functionally equivalent to lambda lysis gene S. Gene S codes for two products, the polypeptides S105 and S107, produced from translational initiation events at the third and first codon, respectively. We have shown that the two polypeptides have opposing functions in lysis: S105 is the lethal lysis effector, and S107 acts as an inhibitor of lysis (U. Blasi, K. Nam, D. Hartz, L. Gold, and R. Young, EMBO J. 11:3501-3510, 1989). Gene 13 has a 108-codon reading frame and its product begins with a similar motif: Met-1-Lys-2-Lys-3-Met-4. Here, we present in vivo and in vitro evidence for the expression of a 13(108) and a 13(105) product and show that the lambda lysis control mechanisms is evolutionarily conserved in phage P22. In this case 13(108), like S107 in lambda, functions as the inhibitor of the lysis effector 13(105). Although the DNA sequences upstream of the S and 13 gene starts showed less homology, the same structural characteristics, i.e., stem-loop structures immediately upstream and about 10 codons downstream of the start region, were present in both reading frames. Using in vitro mutagenesis and toeprinting, we show that the upstream stem-loop structures of genes 13 and S, containing the Shine-Dalgarno sequence for initiations at Met-1, are interchangeable. Moreover, our data indicate that the stability of the secondary structures present in the translational initiation regions of genes S and 13 is set to create a particular ratio of initiation events at Met-1 and Met-3 or Met-4. The ratio of effector to inhibitor was much higher in P22 than in lambda. We propose that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.

Journal ArticleDOI
01 Sep 1990-Gene
TL;DR: An efficient system to control the expression of cloned genes in Bacillus subtilis was established by introducing the Escherichia coli bacteriophage lambda cI857 repressor-pR promoter system into this host and constitutively expressed a staphylokinase reporter gene which was fused to the lambda pR promoter.

Journal ArticleDOI
TL;DR: In this cell-free system, plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target was used and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules.

Journal ArticleDOI
TL;DR: The hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways is proposed.
Abstract: Expression of the red + and gam + genes of bacteriophage λ in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these λ functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2′-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red + or gam + expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam + or red + reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (σ-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red + products, β protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD− Exol− cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3′ singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.

Journal ArticleDOI
TL;DR: Observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.
Abstract: The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.

Journal ArticleDOI
TL;DR: The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.
Abstract: Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.

Journal ArticleDOI
30 Mar 1990-Gene
TL;DR: A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli.


Journal ArticleDOI
TL;DR: Using relatively large glass particles and cells solubilized with guanidine thiocyanate, a 45 min procedure is developed which routinely yields 5—6 fig of highly pure DNA/10 cells from multiple samples.
Abstract: Extraction of DNA from animal cells or tissues with organic solvents yields only 1—2 /tg/10 cells and is labor intensive and time consuming, requiring several hours or days. The affinity of DNA for glass has been exploited to extract DNA from agarose (1) and plasmid from bacterial lysates (2), bt has not been successfully applied to animal DNA extraction because of DNA fragmentation and inefficiency of DNA binding in the presence of cell lysates. By using relatively large glass particles (5—25 y.) and cells solubilized with guanidine thiocyanate, we have been able to develop a 45 min procedure which routinely yields 5—6 fig of highly pure DNA/10 cells from multiple samples. Pelleted cells were first lysed with 5M guanidine thiocyanate/0.1 M EDTA (pH8.0) to a concentration of 2xlO cells per ml. Lysate (100 /xl) was then combined with 500 yX of glass powder (Schleicher and Schuell, Keen, NH) suspended in binding buffer (50 mg glass/ml in 6 M sodium perchlorate, 50 mM Tris-Cl pH 8.0, 10 mM trans-1,2-diaminocyclohexane -N, N, N', N'-tetracetic acid, CDTA), and the suspensions were mixed by rocking at room temperature for 20 min. The glass was pelleted by spinning in a microfuge for 30 sec, washed with 500 /tl of binding buffer, pelleted, then resuspended in 500 /tl of elution buffer (0.2 M sodium perchlorate, 50 mM Tris-Cl pH 8.0, 10 mM CDTA) and rocked at room temperature for 20 min. The glass was then pelleted, and supernatants containing the eluted DNA were transferred to fresh tubes. Two volumes (1 ml) of 100% ethanol were added, the contents were mixed by inverting, and the tubes were spun in a microfuge for 5 min at room temperature. DNA pellets were rinsed with 500 fd of 90% ethanol, dried, then resuspended in restriction endonuclease buffer. In figure 1, lane 'M' contains 1 ng of lambda DNA cleaved with HindHl and EcoKl (Promega). Lanes ' C contain 5 fig of DNA purified by conventional means from spleen tissue (3). Lanes ' 1 ' contain DNA extracted from lysate of 10 K562 cells. All DNAs were incubated for 16 h at 37°C in the absence Qanes '0') or presence (lanes 'X' of 25 units of Xba\\ (Promega). Alternatively, glass was washed once with 50% ethanol and DNA was eluted in any convenient buffer, but yields were reduced 40% (not shown). Bands in the cut lanes are cleavage products from repeat elements in the human genome (3).