The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Deletion of one nucleotide within the homonucleotide tract present in the hsdS gene alters the DNA sequence specificity of type I restriction-modification system NgoAV

As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS(NgoAV1) and hsdS(NgoAV2). The specificity subunit, HsdS(NgoAV), the product of the hsdS(NgoAV1) gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdS(NgoAV1) gene makes the NgoAV system a strong candidate for phase variation, i.e., stochastic addition or reduction in the guanine number. We have constructed mutants with 6 guanines instead of 7 and demonstrated that the deletion of a single nucleotide within the 3' end of the hsdS(NgoAV1) gene restored the fusion between the hsdS(NgoAV1) and hsdS(NgoAV2) genes. We have demonstrated that such a contraction of the homonucleotide tract may occur in vivo: in a Neisseria gonorrhoeae population, a minor subpopulation of cells appeared to have only 6 guanines at the 3' end of the hsdS(NgoAV1) gene. Escherichia coli cells carrying the fused gene and expressing the NgoAVΔ RM system were able to restrict λ phage at a level comparable to that for the wild-type NgoAV system. NgoAV recognizes the quasipalindromic interrupted sequence 5'-GCA(N(8))TGC-3' and methylates both strands. NgoAVΔ recognizes DNA sequences 5'-GCA(N(7))GTCA-3' and 5'-GCA(N(7))CTCA-3', although the latter sequence is methylated only on the complementary strand within the 5'-CTCA-3' region of the second recognition target sequence.     

A chromosomal mutation mediating increased expression of pyrE in Salmonella typhimurium is located within the proposed attenuator

A chromosomal mutation resulting in 15- to 20-fold increased expression of the Salmonella typhimurium pyrE gene has been cloned and sequenced. The mutation was found to be a single base-pair transition of GC to AT, and occurred within a region of dyad symmetry (attenuator) located just upstream of the pyrE structural gene.     

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.     

Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich.

The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mammalian temperature-sensitive mutation affecting G1 progression results from a single amino acid substitution in asparagine synthetase.

ts11 is a temperature-sensitive (ts) mutant isolated from the BHK-21 Syrian hamster cell line that is blocked in the G1 phase of the cell cycle at the non-permissive temperature (39.5 degrees C). We previously showed that the human gene encoding asparagine synthetase (AS) transformed ts11 cells to a ts+ phenotype and that ts11 cells were auxotrophic for asparagine at 39.5 degrees C. We show here that ts11 cells exhibit a ts phenotype for AS activity, and that the ts11 AS was much heat-labile than the wt enzyme. We have isolated AS cDNAs from wt BHK and ts11 cells and found that wt, but not ts11 AS cDNAs were capable of transformation. The deduced amino acid sequence of Syrian hamster AS showed 95% identity to the human protein as well as the same number of residues. The inability of the ts11 AS cDNAs to transform was due to a single base change, a C to T transition, that would result in the substitution of leucine with phenylalanine at a residue located in the C-terminal fourth of the enzyme. Thus the ts11 mutation identifies a mutated, thermolabile AS.     

Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins.

Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.     

The araIc mutation in Escherichia coli B/r.

The araIc allele is a cis-acting mutation which has been used to define the araBAD promoter in Escherichia coli B/r. Nineteen araIc mutants were originally isolated by Englesberg and co-workers as Ara+ "revertants" of an araC deletion mutant (Englesberg et al. J. Mol. Biol. 43:281-298, 1969). The mutants constitutively expressed araBAD gene products in the absence of functional araC activator protein. Eight of the araIc mutations have been cloned by in vivo recombination onto pBR322-ara hybrid plasmids. Restriction and DNA sequence analysis of these araIc mutations showed that they result from a single base-pair change located at -35 in the araBAD promoter.     

Sequence-dependent effects on DNA stability resulting from guanosine replacements by inosine.

The effect of replacing a G.C base-pair with an I.C base-pair on DNA stability was investigated for a related set of 14-mers. DNA melting analysis of the 14-mers was used to determine delta Hzero, delta Szero and delta G(zero)37 of the double to single stranded transition. All 14mers were shown to have B-DNA character by circular dichroism analysis. 14mers substituted with a single inosine in place of guanosine at different positions showed that consequences on DNA stability are sequence-dependent. Large changes in delta Hzero and delta Szero result when inosine is substituted within the trinucleotide sequence d(TCG).d(CGA) while substitution within d(TCC).d(GGA) causes minor changes in enthalpy and entropy. Moreover, some 14-mers with two inosine substitutions five base-pairs apart showed non-additive free energy changes for the double to single stranded transition. These results clearly indicate that the structural consequences of replacing a single guanosine with an inosine are transmitted over a significant distance.     

Temperature gradient gel electrophoresis: detection of a single base substitution in the cattle β-lactoglobulin gene

An A in equilibrium with G transition in exon III is known to differentiate alleles A and B of the cattle beta-lactoglobulin (BLG) gene. A BLG exon III fragment containing the transition site was amplified by the polymerase chain reaction. Temperature gradient gel electrophoresis (TGGE) was then used to detect this transition and hence to genotype cattle: the AT base-pair in allele A was readily distinguished from the GC base-pair of allele B. TGGE can be used to detect any single base-pair substitution, and thus is a powerful method of detecting genetic variability.     

Partitioning of plasmid R1. Structural and functional analysis of the parA locus

The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.     

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.     

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.     

Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.     

Sequence-specific recognition of DNA. Nuclear magnetic resonance assignments and structural comparison of wild-type and mutant lambda OR3 operator DNA

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.     

Functional characterization of thermolabile DNA-binding proteins that affect adenovirus DNA replication.

The human adenovirus type 2 (Ad2) mutant Ad2ts111 has previously been shown to contain two mutations which result in a complex phenotype. Ad2ts111 contains a single base change in the early region 1B (E1B) 19,000-molecular-weight (19K) coding region which yields a cyt deg phenotype and another defect which maps to the E2A 72K DNA-binding protein (DBP) coding region that causes a temperature-sensitive DNA replication phenotype. Here we report that the defect in the Ad2ts111 DBP is due to a single G----T transversion that results in a substitution of valine for glycine at amino acid 280. A temperature-independent revertant, Ad2ts111R10, was isolated, which reverts back to glycine at amino acid 280 yet retains the cyt and deg phenotypes caused by the 19K mutation. We physically separated the two mutations of Ad2ts111 by constructing a recombinant virus, Ad2ts111A, which contained a wild-type Ad2 E1B 19K gene and the gly----val mutation in the 72K gene. Ad2ts111A was cyt+ deg+, yet it was still defective for DNA replication at the nonpermissive temperature. The Ad2ts111 DBP mutation is located only two amino acids away from the site of the mutation in Ad2+ND1ts23, a previously sequenced DBP mutant. Biochemical studies of purified Ad2+ND1ts23 DBP showed that this protein was defective for elongation but not initiation of replication in a cell-free replication system consisting of purified Ad polymerase, terminal protein precursor, and nuclear factor I. Ad2+ND1ts23 DBP bound less tightly to single-strand DNA than did Ad2 DBP, as shown by salt gradient elution of purified DBPs from denatured DNA cellulose columns. This decreased binding to DNA was probably due to local conformational changes in the protein at a site that is critical for DNA binding rather than to global changes in protein structure, since both the Ad2+ND1ts23 and Ad2 DBPs showed identical cleavage patterns by the protease thermolysin at various temperatures.     

Isolation of a trans-dominant histidase-negative mutant of Salmonella typhimurium

A mutation of Salmonella typhimurium was obtained that results in the failure of cells to synthesize the enzyme l-histidine ammonia-lyase (histidase). The mutation mapped within the hutH gene and in merodiploid strains was dominant over the wild-type allele. Extracts from cells bearing the trans-dominant histidase-negative allele were shown to contain material that reacts immunologically with antiserum against purified wild-type histidase. It is proposed that the trans-dominant allele results in the synthesis of defective histidase subunits that can combine with, and partially inactivate, wild-type histidase subunits. This subunit mixing presumably does occur, as the enzyme synthesized in a hybrid merodiploid strain is abnormally heat sensitive.