Producing a P1 bacteriophage library from pine: isolation and cloning of very high molecular weight DNA.

We have generated a genomic P1 bacteriophage library using Monterey pine (Pinus radiata) DNA. We first developed a method for isolating from pine tissue the very high molecular weight DNA necessary for the preparation of libraries requiring large inserts. The method involves protoplasting the cells, isolating nuclei and lysis in a high concentration of detergent. Fragments of greater than two megabases in size are produced in solution. Modifications introduced to the protocol for library preparation and for P1 plasmid isolation are described.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

Isolation and characterization of the smallest bacteriophage P4 derivatives packaged into P4-size head in bacteriophage P2-P4 system

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.     

A U3 RNA pseudogene in mouse: sequence and organization in genomic DNA

A mouse U3 RNA pseudogene has been identified; it corresponds to a U3B full length coding sequence with a 3'-oligo(A) tail, precisely flanked at both ends by a pair of 15 bp direct repeats. These structural features suggest that it has arisen through an RNA-mediated mechanism involving an insertion at staggered nicks in the genome. Sequence data indicate that this mouse specimen has been generated by a different event as compared to the recently described rat pseudogenes. It represents the first reported case, for a pseudogene of this class, to be present at more than one copy per genome.     

Molecular cloning and characterization of endogenous feline leukemia virus sequences from a cat genomic library.

Recombinant bacteriophage lambda clones from a cat genomic library derived from placental DNA of a specific pathogen-free cat were screened to identify endogenous feline leukemia virus (FeLV) sequences. Restriction endonuclease mapping of four different clones indicates that there are a number of similarities among them, notably the presence of a 6.0- to 6.4-kilobase pair (kbp) EcoRI hybridizing fragment containing portions of sequences homologous to the gag, pol, env, and long terminal repeat-like elements of the infectious FeLV. The endogenous FeLV sequences isolated are approximately 4 kbp in length and are significantly shorter than the cloned infectious FeLV isolates, which are 8.5 to 8.7 kbp in length. The endogenous elements have 3.3- to 3.6-kbp deletions in the gag-pol region and approximately 0.7- to 1.0-kbp deletions in the env region. These deletions would render them incapable of encoding an infectious virus and may therefore be related to the non-inducibility of FeLV from uninfected cat cells and the subgenomic expression of these endogenous sequences in placental tissue. It appears that there is conservation in the ordering of restriction sites previously reported in the proviruses of the infectious FeLVs in sequences corresponding to the pol and env boundary as well as the region spanning the env gene of the endogenous clones, whereas a greater divergence occurs among restriction sites mapped to the gag and part of the pol regions of the infectious FeLV. Such deleted, FeLV-related subsets of DNA sequences could have originated either by germ-line integration of a complete ecotropic virus followed by deletion, or by integration of a preexisting, defective, deleted variant of the infectious virus.     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.