The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.

Temperature-sensitive mutants of the EcoRI endonuclease.

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro.

Patenting cDNA 1993: efforts and happenings (Human Genome Project; NIH: US PTO; onco-mouse; European Patents).

The National Institute of Health (NIH) and Dr. J.C.

In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog.

We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro. The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation. Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo.

Translational repression in bacteriophage f1: characterization of the gene V protein target on the gene II mRNA.

Previous studies have shown that the single-stranded DNA binding protein of bacteriophage f1 (gene V protein) represses the translation of the mRNA of the phage-encoded replication protein (gene II protein). We have characterized phage mutations in the repressor and in its target. Using a gene II-lacZ translational fusion, we have defined a 16-nucleotide-long region in the gene II mRNA sequence that is required in vivo for repression by the gene V protein.

Repair of the Escherichia coli chromosome after in vivo scission by the EcoRI endonuclease.

We prepared a set of temperature-sensitive mutants of the EcoRI endonuclease. Under semipermissive conditions, Escherichia coli strains bearing these alleles form poorly growing colonies in which intracellular substrates are cleaved at EcoRI sites and the SOS DNA repair response is induced. Strains defective in SOS induction (lexA3 mutant) or SOS induction and recombination (recA56 and recB21 mutants) are not more sensitive to this in vivo DNA scission, whereas strains deficient in DNA ligase (lig4 and lig ts7 mutants) are extremely sensitive.

Integration host factor interacts with the DNA replication enhancer of filamentous phage f1.

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria.

A hybrid toxin from bacteriophage f1 attachment protein and colicin E3 has altered cell receptor specificity.

A hybrid protein was constructed in vitro which consists of the first 372 amino acids of the attachment (gene III) protein of filamentous bacteriophage f1 fused, in frame, to the carboxy-terminal catalytic domain of colicin E3. The hybrid toxin killed cells that had the F-pilus receptor for phage f1 but not F- cells. The activity of the hybrid protein was not dependent upon the presence of the colicin E3 receptor, BtuB protein.

Hemimethylation prevents DNA replication in E. coli.

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication.

Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein.

Initiation of DNA synthesis at an origin of DNA replication involves complex protein-DNA interactions that are still poorly understood. Some of these interactions are highly specific and involve proteins (initiator proteins) thought to be essential for regulation of the initiation process because of their rate-limiting activity. We show here that both qualitative and quantitative changes in one of these proteins have profound effects on protein-DNA interactions at an origin of DNA replication, and are sufficient to reduce to less than one-third the minimal sequence required for initiation.

Increased intracellular concentration of an initiator protein markedly reduces the minimal sequence required for initiation of DNA synthesis.

One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication.

The functional origin of bacteriophage f1 DNA replication. Its signals and domains.

The origin of DNA replication of bacteriophage f1 functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage f1 led us to postulate the existence of a set of overlapping functional domains.

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells.

The origin of DNA replication of bacteriophage f1 and its interaction with the phage gene II protein.

The origin of DNA replication of bacteriophage f1 consists of two functional domains: 1) a "core region", about 40 nucleotides long, that is absolutely required for viral (plus) strand replication and contains three distinct but partially overlapping signals, a) the recognition sequence for the viral gene II protein, which is necessary for both initiation and termination of viral strand synthesis, b) the termination signal, which extends for 8 more nucleotides on the 5' side of the gene II protein recognition sequence, c) the initiation signal that extends for about 10 more nucleotides on the 3' side of the gene II protein recognition sequence; 2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the "secondary region" does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). This region, however, can be made entirely dispensable by mutations elsewhere in the phage genome.

The morphogenetic signal of bacteriophage f1.

The genome of the single-stranded DNA phage f1 contains an intergenic region (IG), 508-nucleotides long, that does not code for any known protein. By use of a system of chimeric plasmids haboring different f1 fragments, we had previously shown that this region contains, in addition to the f1 'functional origin' of DNA replication, a signal of less than 300 nucleotides required for efficient morphogenesis to occur ('morphogenetic signal'). In the present study, we have localized this signal to within a sequence of less that 60 nucleotides of almost perfect palindromic symmetry at the genet IV/IG border.