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A recombinant lambda bacteriophage isolated from a human genome library contains the gene for fibroblast interferon (IFN-beta1). The DNA sequence of this gene is identical to the sequence of its mRNA and is devoid of introns.
A single recombinant lambda bacteriophage isolated from a human genome library contains two closely related human interferon genes of the leukocyte or alpha type. The two genes are separated by 12 kilobase pairs and are oriented in the same direction with respect to transcription. Comparisons of the DNA sequences of these two genes and interferon complementary DNA clones indicate that the two interferon genes lack intervening sequences.
The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.
The construction of three lambda bacteriophages containing parts of the structural gene for threonyl-tRNA synthetase, thrS, and those for the two subunits of phenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages were used as hybridization probes to measure the in vivo levels of mRNA specific to these three genes. Plasmid pB1 carries the three genes thrS, pheS, and pheT, and strains carrying the plasmid show enhanced levels of mRNA corresponding to these genes. Although the steady-state levels of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase produced by the presence of the plasmid differed by a factor of 10, their pulse-labeled mRNA levels were about the same. Mutant derivatives of pB1 were also analyzed. Firstly, a cis-acting insertion located before the structural genes for phenylalanyl-tRNA synthetase caused a major decrease in both pheS and pheT mRNA. Secondly, mutations affecting either structural gene pheS or pheT caused a reduction in the mRNA levels for both pheS and pheT. This observation suggests that autoregulation plays a role in the expression of phenylalanyl-tRNA synthetase.
A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.
Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.
To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences. We found a Ter site, which we named TerE, located at 23.2 min on the linkage map. TerE inhibits only counterclockwise DNA replication. Macroscopically, five Ter sites are located in a periodic arrangement on the genome.
We isolated two clones of recombinant lambda bacteriophage with overlapping inserts of Bacillus subtilis chromosomal DNA corresponding to part of the flaA locus. The flaA4 and flaA15 mutations were localized on the physical map by marker rescue experiments. The flaA locus and the flaB (sigD) gene were mapped in transduction crosses, and the order glnA polC flaB flaA was determined. FlaB was linked to polC in transformation crosses.
Using low-stringency Southern blot analysis and cloning in lambda bacteriophage, two new human papillomavirus types (HPV-43 and HPV-44) were identified and their DNAs were cloned from vulvar tissues. The isolates were characterized by restriction endonuclease mapping and shown to be new HPV types on the basis of their minimal hybridization with all other known HPV types at high stringency. Both HPVs are most closely related to types 6, 11, and 13. HPV-43 did not exhibit any cross-reactivity with these HPV types at high stringency. HPV-44 showed minimal cross-reactivity to HPV-13, which was in the range of 20 to 25% according to liquid hybridization analysis. The deduced genomic organization of each of the two new HPVs was colinear with HPV-6b. Prevalence studies revealed that HPV-43 and HPV-44 together were found in 6 of 439 normal cervical tissues, in 8 of 195 cervical intraepithelial neoplasms, but in none of 56 cervical cancers tested thus far.
We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.
The interaction of three factors determine the detrimental effect of a palindromic DNA sequence in Escherichia coli cells. The first is the nature of the palindrome (its length, extent of central asymmetry and perhaps its base sequence), the second is the genotype of the host cell and the third is the replicon within which it is located. In this paper we extend the genetic and physical characterization of lambda bacteriophages carrying a palindrome of approximately 560 base pairs. We also show that a palindrome of approximately 110 base pairs which can be cloned in a plasmid cannot be cloned in a phage M13 derivative. These observations are relevant to the choice of vectors used in the cloning of eukaryotic DNA containing palindromic sequences.
Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.
The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol. Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method. The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation. The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium. The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing. Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction. Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.
The intramolecular deletion-generating recombination which transforms lambda bacteriophage genomes into the plasmids (named pLS) proved to be site-specific to a certain extent. Using electron microscopy heteroduplex analysis three preferential sites for this recombination were found in seven independent pLS isolates studied. Att-sites were not registered to be involved in the formation of deletions in isolates studied. It was shown that recombination operating in our system was independent of the phage int and bacterial recA genes.
The recombinant plasmid pKP145 PL has been constructed containing the gene for aminoglycosidephosphotransferase (APT). Expression of the APT gene is under the control of lambda bacteriophage PL promoter. Escherichia coli cells harbouring this plasmid synthesize APT in quantity up to 13-15% of the total cellular protein. The technique for isolation of APT from superproducing cells has been elaborated. Preparations of the enzyme devoid of contaminating bacterial proteins have been obtained.
An indirect enzyme-linked immunosorbent assay (ELISA) has been described to detect and measure the antibodies in O. mykiss against a lambda bacteriophage antigen (strain c1187 Sam7). Antibodies were first detected six days after the first intraperitoneal injection and by day 21 all fish were positive. Maximum mean titres were detected on day 30 (3 days after the second injection). The mean titre declined by day 33 which was 6 days after second injection and remained constant from day 39 to day 45. The results suggest that immune response in a rainbow trout occurred faster and the fish were able to mount a very good response to viruses.
A genomic lambda gt11 DNA library of Mycobacterium tuberculosis was screened for expression of mycobacterial protein antigens with murine monoclonal antibodies. The reactivity patterns of the monoclonal antibodies ranged from those showing a limited interspecies reactivity to antibodies widely cross-reactive among different mycobacterial species. Twelve recombinant bacteriophages were isolated, containing eight mycobacterial genes (paa, pab, pac, pad, paeA, paeB, pafA, and pafB) encoding protein antigens. Physical maps of the phages were generated and the products of the recombinant genes were analyzed by immunoblotting techniques. PaeA and PaeB are distinct proteins but were shown to share an epitope. A similar condition was observed between PafA and PafB. Among the phages isolated, two groups expressed epitopes specific for M. tuberculosis and Mycobacterium bovis BCG. One group of phages produced an antigenic determinant which is found in M. tuberculosis and Mycobacterium marinum but not in M. bovis BCG.
A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.