PubMed Health⌕ Search

Biomedical subjects

L W Black

Publications and source records attributed to L W Black.

15 recordsLinked to original sources
PubMed1992

A phage T4 in vitro packaging system for cloning long DNA molecules.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.

Bacteriophage lambda↗
PubMed1978

Purification and properties of the bacteriophage T4 gene 31 protein required for prehead assembly.

A low molecular weight (approximately 16,000), early protein is characterized as the product of the essential T4 head assembly gene 31. This gene is known to be required to allow formation of any ordered head structure from the major T4 capsid protein, P23 (Laemmli, U.K., Beguin, F., and Gujer-Kellenberger, G. (1970) J. Mol. Biol. 47, 69-85). In wild type infection P31 synthesis ceases at late times; in contrast, P31 is overproduced in certain early or regulatory T4 mutant infections, particularly gene 55 mutant infections. P31 was purified preparatively from Escherichia coli infected with the latter mutant, but could only be obtained for the most part in modified form, possibly due to unusual sensitivity to a proteolytic activity. P31 is not cleaved in vivo during normal head assembly, nor does it become a part of the mature head or any ordered prehead structure as determined by an immunological assay using antiserum prepared against the purified protein. However P31 does appear to become a part of the unordered P23 aggregates (lumps) which accumulate when ordered P23 assembly is prevented. We cound find no evidence for P31 association with T4 DNA or the host membrane. Our experiments favor the hypothesis that P31 directly affects the aggregation state and solubility properties of P23.

Amino Acids↗
PubMed1978

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.

Coliphages↗
PubMed1977

DNA packaging and the pathway of bacteriophage T4 head assembly.

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction.

Coliphages↗
PubMed1976

Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.

Amino Acid Sequence↗
PubMed1976

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).

Coliphages↗