rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

A chain of interlinked genes in the ninR region of bacteriophage lambda

The 3612-bp DNA sequence of the phage lambda P-Q (ninR) region contains a series of nine open reading frames in a distinctly overlapping pattern: ATGA sequence modules occur at the boundaries of consecutive genes and are able to serve both as terminator (TGA) and (re)initiator (ATG) codons for most of the adjacent frames. Together with genes O, P, and Q, the newly detected ren and ninA through ninH constitute a series of twelve closely linked genes in the pR operon. Based upon the available evidence for several of the nin proteins, and on plasmid expression data, we conclude that at least the larger nin genes, and probably all of the newly detected open reading frames code for proteins. The nin5 deletion of 2803 bp is a frame-to-frame fusion of ren and ninH, and covers the t R2 termination signal located near its left boundary, immediately behind the ren gene. The possible significance of the observed chain of closely interlinked genes for the regulation of Q expression is discussed.     

An Mr 29000 protein is essential for mini-F maintenance in E. coli

Plasmids consisting of mini-F inserted into multicopy vectors were constructed. Derivatives of these hybrid replicons were isolated which contained the transposon Tn5. The polypeptides encoded by these plasmids were identified by Escherichia coli minicell analysis. We show that a previously unidentified polypeptide of 29000 Mr is encoded by the mini-F gene E between 45.1 and 46.2 F kb on the mini-F plasmid map, and that this coding sequence (E gene) is transcribed rightward. Hybrid plasmids carrying Tn5 inserted into the E gene are unable to replicate in a polA- strain. Hence the E protein is essential for mini-F replication. Mutations in the A and B genes of mini-F affect E gene expression, and the results suggest that E protein synthesis is stimulated by A protein.     

The nucleotide sequence of the yeast ARG4 gene

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated M_r of 52010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.     

The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

Differential binding of RNA polymerase to the pRM and pR promoters of bacteriophage lambda

Escherichia coli RNA polymerase binding to the promoters pR and pRM of bacteriophage lambda was visualized and quantitated by electron microscopy. Although the two promoters are located close together in the phage genome, their proximity to the end of an 889-bp HaeIII DNA fragment made it possible to position binary complexes within 18 bp (2%) intervals. Thus, polymerase binding to pR and pRM could be distinguished by comparing the locations of binary complexes formed with wild-type and mutant (prm-) DNA at 37 degrees and 15 degrees C. We found that at 37 degrees C, RNA polymerase bound primarily to pR, while at 15 degrees C the efficiency of binding was the same at pRM as at pR. In addition, at 15 degrees C the overall efficiency of binding was significantly reduced relative to that at 37 degrees C. When the enzyme was incubated with prm- DNA, binding to pRM was reduced at both temperatures, as expected. Reduced binding to pRM was accompanied by an increase in binding to pR, apparently as a consequence of the low enzyme-to-DNA ratios used in these experiments.     

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.     

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Cloning of the replication gene O of E. coli bacteriophage lambda and its expression under the control of the lac promoter

The expression of the replication gene O of bacteriophage lambda was put under the control of the lac promoter-operator region integrated into the pBR322 cloning vehicle. The new plasmid pKK104 was introduced into minicells and the O gene induced by isopropyl-beta-thiogalactoside (IPTG). The O protein could be identified as a major component in extracts from these cells, in association with the cell membrane fractions. The molecular weight of the O protein in SDS gels is about 33 000, and it is metabolically unstable but apparently stable upon isolation as a membrane-associated fraction.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

Restriction endonuclease analysis of DNA from the Streptomyces phages SH3, SH5, SH10 and SH13

Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only EcoRI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by EcoRI, KpnI and BglII, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clearplaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two KpnI sites.     

Complete nucleotide sequence of a chicken α-globin cDNA

The entire sequence of a 541 bp insert in recombinant plasmid pHb1003 has been determined. This plasmid, which was shown to carry a cloned cDNA copy of the chicken α-globin mRNA, contains the complete structural gene as well as 19 bp of the 5'-untranslated region and 99 bp of the 3'-untranslated region. This sequence may encode a non-adult α-globin gene, especially since the cDNA clones were generated from phenylhydrazine-induced, globin-specific mRNA extracted from anemic white leghorns. The possibility that this α-globin might represent a stress globin is considered.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

The DNA between Rz and cosR in bacteriophage lambda is nonessential

Near the right end of phage lambda DNA, between gene Rz and the cos site, are 2050 bp of apparently non-coding DNA. We have cloned a lambda DNA fragment containing this DNA into a plasmid and constructed a deletion, omega l, extending from a site within the Rz gene to a site about 560 bp from cos. This deletion could be recombined into viable lambda phage at a frequency equal to that observed for the undeleted sequence. Recombinant phage lambda carrying the omega l deletion were demonstrated to have the same burst size and kinetics of phage production as undeleted lambda. The omega l deletion can be used to extend the capacity of lambda cloning vectors and to provide a region for the insertion of heterologous DNA which should exhibit controllable high level expression from the lambda late promoter, p'R.     

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.     

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA

P450_scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450_scc indicates P450_scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the $\text{PstI}$ site of pBR322 by dC·dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450_scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.