Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system

The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors.     

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.     

Marker rescue studies of the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 x 10(-6) to 5.8 x 10(-7) transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 x 10(-9)). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 x 10(-10)), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 x 10(-11). No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

Extrachromosomal Elements in Group N Streptococci

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.     

Heterogeneity of deoxyribonucleic acid molecules isolated from Mycobacterium smegmatis.

Bulk chromosomal deoxyribonucleic acids (DNAs) of Mycobacterium smegmatis strains 607+ (wild type) and 607-1 (Strr) and orange-red pigmented variants (OR) were separated into two distinct bands (types 1 and 2) by cesium chloride density gradient centrifugation. Thermal denaturation analyses showed that type 1 and 2 DNA fragments of these strains possessed guanine plus cytosine contents averaging 69.2% and 60.8%, respectively. Type 1 and 2 DNAs from all strains tested were recovered in relatively equal quantities upon isolation and were found to have similar molecular weights (3.0 x 10(7)). Spectrophotometric assay of DNA reassociation showed that homology between any type 1 and 2 DNA fragments was always very low (29 to 33%), even within the same strain. Homologies among type 1 DNAs isolated from any strain were always high (92 to 98%), whereas homologies between type 2 DNA isolated from OR strains and that from their parental strain 607-1 were lower (51 to 55%). Transformation experiments revealed that methionine, leucine, folic acid, and streptomycin markers were found exclusively in type 1 DNA fragments. In addition to the two types of chromosomal DNA, plasmid DNA possessing a molecular weight of about 4 x 10(6) was found in strain 607-1.     

Virulence conversion of Legionella pneumophila by conjugal transfer of chromosomal DNA

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.     

Replacement recombination in Lactococcus lactis.

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid. Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.     

Mutagenic radioadaptation in a human lymphoblastoid cell line

We investigated the mutagenic radioadaptive response of human lymphoblastoid TK6 cells by pretreating them with a low dose (5 cGy) of X-rays followed by a high (2 Gy) dose 6h later. Pretreatment reduced the 2-Gy-induced mutation frequency (MF) of the thymidine kinase (TK) gene (18.3 x 10(-6)) to 62% of the original level (11.4 x 10(-6)). A loss of heterozygosity (LOH) detection analysis applied to the isolated TK(-) mutants revealed the mutational events as non-LOH (resulting mostly from a point mutation in the TK gene), hemizygous LOH (resulting from a chromosomal deletion), or homozygous LOH (resulting from homologous recombination (HR) between chromosomes). For non-LOH events, pretreatment decreased the frequency to 27% of the original level (from 7.1 x 10(-6) to 1.9 x 10(-6)). cDNAs prepared from the non-LOH mutants revealed that the decrease was due mainly to the repression of base substitutions. The frequency of hemizygous LOH events, however, was not significantly altered by pretreatment. Mapping analysis of chromosome 17 demonstrated that the distribution and the extent of hemizygous LOH events were also not significantly influenced by pretreatment. For homozygous LOH events, pretreatment reduced the frequency to 61% of the original level (from 5.1 x 10(-6) to 3.1 x 10(-6)), reflecting an enhancement in HR repair of DNA double-strand breaks. Our findings suggest that the radioadaptive response in TK6 cells follows mainly from mutations at the base-sequence level, not the chromosome level.     

Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis

Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.     

Colinearity between the DNAs of Epstein-Barr virus and herpesvirus papio.

Epstein Barr virus (EBV) and herpesvirus papio (HVPapio) DNAs share a common format and 40% homology. Labeled cloned fragments of EBV DNA were hybridized to blots of XbaI, EcoRI, HindIII, and SalI fragments of HVPapio DNA. EBV fragments which mapped from 2 x 10(6) to 54 x 10(6) and from 59 x 10(6) to 106 x 10(6) daltons hybridized to fragments at identical map positions in HVPapio DNA. Regions of nonhomology were demonstrated at 0 x 10(6) to 2 x 10(6), 54 x 10(6) to 59 10(6), and 106 x 10(6) to 115 x 10(6) daltons.     

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.     

Characterization of nuclear DNA in 12 species of Chlorella (Chlorococcales, Chlorophyta) by DNA reassociation

Strains of 12 different species of the genus Chlorella were analyzed for amount, reiteration frequency and kinetic complexity of chromosomal DNA components by C0t analysis. The resulting C0t curves reveal at least two different DNA components consisting of single copy DNA (up to 95%) and of repetitive DNA with complexities of 4.1 x 10(3) base pairs (bp) to approximately 11.7 x 10(3) bp and a reiteration frequency of 100-760. The total amount of repetitive DNA is less than 9% of the nuclear genome and similar in all strains studied. In contrast, the total kinetic complexity varies in a wide range from 1.26 x 10(7) bp to 8.08 x 10(7) bp which is mainly due to differences in the size of single copy DNA. The genome sizes in Chlorella seem not to be correlated with biochemical and physiological characteristics and therefore are unlikely to be useful as a taxonomical marker. A comparison of thermal denaturation profiles showed that the melting points of repetitive and single copy DNA differ by approximately 7 degrees C which may result from base mismatch and/or from a distinct base composition of the repetitive DNA.     

DNA of herpesvirus pan, a third member of the Epstein-Barr virus-Herpesvirus papio group

The DNA of herpesvirus pan, a primate B-lymphotropic herpesvirus, shares about 40% well-conserved sequence relatedness with Epstein-Barr virus (EBV) and herpesvirus papio DNAs. Labeled cloned fragments from the EBV recombinant DNA library were cross hybridized to blots of EcoRI, XbaI, and BamHI restriction endonuclease fragments of herpesvirus pan DNA to identify and map homologous sequences in the herpesvirus pan genome. Regions of colinear homology were demonstrated between 6 x 10(6) daltons and 108 x 10(6) daltons in the DNAs. The structural organization of herpesvirus pan DNA was similar to the format of Epstein-Barr virus and herpesvirus papio DNAs. The DNA consists of two domains of largely unique sequence complexity, a segment US of 9 x 10(6) daltons and a segment UL of 88 x 10(6) daltons. US and UL are separated by a variable number of tandem repetitions of a sequence IR (2 x 10(6) daltons). There was homology between DNA which mapped at 26 to 28 x 10(6) daltons and 93 to 95 x 10(6) daltons in UL. The terminal reiteration component, TR, of herpesvirus pan DNA and sequences which mapped to the left of 6 x 10(6) daltons and to the right of 108 x 10(6) daltons had no detectable homology with the corresponding regions of Epstein-Barr virus DNA.     

Molecular Structure of an R Factor, Its Component Drug-Resistance Determinants and Transfer Factor

Plasmid DNA from Escherichia coli strains harboring drug resistance either of the infectious or noninfectious kind has been separated by CsCl centrifugation of crude cell lysates in the presence of ethidium bromide and examined by electron microscopy. Plasmid deoxyribonucleic acid (DNA) from an S(+) strain (which has the property of noninfectious streptomycin-sulfonamide resistance) consists of a monomolecular covalently closed circular species of 2.7 mum in contour length (5.6 x 10(6) atomic mass units; amu). DNA from a strain carrying a transfer factor, termed Delta, but no determinant for drug resistance, is a monomolecular covalently closed circular species of 29.3 mum in contour length (61 x 10(6) amu). DNA from either Delta(+)A(+) or Delta(+)S(+) strains, (which are respectively ampicillin or streptomycin-sulfonamide resistant, and can transfer this drug resistance), shows a bimodal distribution of molecules of contour lengths 2.7 mum and 29.3 mum, whereas DNA from a (Delta-T)(+) strain (showing infectious tetracycline resistance) contains only one species of molecule measuring 32.3 mum (67 x 10(6) amu). We conclude that ampicillin resistance is carried by a DNA molecule (the A determinant) of 2.7 mum, and streptomycin-sulfonamide resistance is carried by an independent molecule (the S determinant) of similar size. These molecules are not able to effect their own transfer, but can be transmitted to other cells due to the simultaneous presence of the transfer factor, Delta, which also constitutes an independent molecule, of size 29.3 mum. In general, there appears to be little recombination or integration of the A or S molecules into that of Delta, although a small proportion (5-10%) of recombinant molecules cannot be excluded. In contrast, the third drug-resistance determinant, that for tetracycline resistance (denoted as T), is integrated in the Delta molecule to form the composite structure Delta-T of size 32.3 mum, which determines infectious tetracycline resistance. The Delta(+)A(+) and Delta(+)S(+) strains are defined as harboring plasmid aggregates, and the (Delta-T)(+) strain is defined as carrying a plasmid cointegrate; the properties of all three strains are characteristic of strains harboring R factors. These results are compatible with the previously published genetic data. The number of Delta molecules per cell appears to be equal to the chromosomal number irrespective of growth phase, and this plasmid can thus be defined as stringently regulated in DNA replication. In contrast, S and A exist as multiple copies, probably in at least a 10-fold excess of chromosomal copy number. S and A can thus be defined as relaxed in the regulation of their DNA replication.     

Transformation and Deoxyribonucleic Acid Size: Extent of Degradation on Entry Varies with Size of Donor

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.     

Transduction of R Factors by a Proteus mirabilis Bacteriophage

A transducing phage, designated phim, was isolated from a lysogenic strain of Proteus mirabilis and was characterized with respect to its physical and genetic properties. The phage contains double-stranded deoxyribonucleic acid (DNA) with an S(20,w) degrees of 29 which corresponds to a molecular weight of 24 x 10(6) daltons. The base composition of phim DNA was estimated to be 40% guanine plus cytosine on the basis of the buoyant density of the DNA. phim carries out generalized transduction of chromosomal genes in P. mirabilis at a frequency of 5 x 10(-8) to 2 x 10(-6) per adsorbed phage. To obtain R-factor transduction, it was necessary to have a resident R factor in the recipient cells. In these experiments, different combinations of genetically distinguishable R factors were used in the donor and recipient cells. The frequencies of R-factor transduction were 10(-9) to 2 x 10(-8). The transduction of R factors using an R(-) recipient could not be detected. Transductant R factors were usually recombinant between donor and resident R factors. All of the transduced R factors were transferable by conjugation. A plausible explanation for the requirement for a resident R factor in the recipient cells is that phim transduces only a portion of the R-factor genome and therefore requires a resident R factor for genetic recombination. The reason for the low frequencies of R-factor transduction is not known, but some possible interpretations have been discussed.     

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.     

Human cytomegalovirus genome: partial denaturation map and organization of genome sequences.

Contour-length measurements of both nondenatured and partially denatured DNA from purified extracellular human cytomegalovirus indicate that more than one size class of viral DNA is encapsidated. In addition to a size class averaging about 100 x 10(6) daltons, a much less abundant class of larger viral DNA molecules, 150 x 10(6) to 155 x 10(6) daltons, was extracted from purified extracellular virus. As predicted by melting-curve analysis, partial denaturation of human cytomegalovirus DNA generates denaturation maps showing distinctive adenine plus thymidine (A+T)-rich and guanine plus cytosine (G+C)-rich localizations. Alignment of partial denaturation maps of both 100 x 10(6)- and 150 x 10(6)- to 155 x 10(6)-dalton molecules from maximum overlap of common A+T- and G+C-rich zones clearly shows six unique zones contained in a length equal to the longest class, 150 x 10(6) to 155 x 10(6) daltons. However, various alignments of the smaller class of the molecules within the confines of the approximately 100 x 10(6)-dalton-length equivalent are nondistinctive. Of the six unique A+T- and G+C-rich zones, five are linked in a specific sequence and maintain the same relative orientation; these features indicate the absence of major inversions within these zones. The sixth unique zone may occur at either end of this five-zone series, but it was never found at both ends of the same molecule. Additionally, this terminal zone appears to undergo complete inversions at least at one end of the alignment, and perhaps at both. These data indicate that 150 x 10(6)- to 155 x 10(6)-dalton molecules comprise human cytomegalovirus-specific genetic information.     

Purification of a DNA-binding protein from Xenopus laevis unfertilized eggs.

A DNA-binding protein from Xenopus laevis unfertilized eggs has been purified to apparent homogeneity. It is a heat stable, lysine-rich protein and has a molecular weight corresponding to 8,200 daltons, measured by sodium dodecyl sulphate gel electrophoresis. The protein, which is active in a monomeric form, stimulates DNA polymerase alpha, and binds to single and double stranded DNA. One egg contains about 4 x 10(12) molecules (minimum estimate) of the protein; since we calculate that 4 x 10(8) molecules are sufficient to cover the entire genome (haploid complement), there is much more protein than is needed to cover chromosomal DNA.