Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.     

Sequence of a secondary phage lambda attachment site located between the pentitol operons of Klebsiella aerogenes.

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.     

Active site localization in a viral mRNA capping enzyme

Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.     

Isolation and characterization of the Aspergillus niger trpC gene

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.     

Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin.

cDNA clones for vitronectin, a cell adhesion-promoting plasma and tissue protein, were isolated from a lambda gt11 library containing cDNA inserts made from human liver mRNA. The library was screened with anti-vitronectin antibodies and the positive clones were further identified with synthetic oligonucleotide probes deduced from the partial amino acid sequence of vitronectin. Nucleotide sequence analysis showed that the largest insert was 1545 bp long and contained the whole sequence corresponding to plasma vitronectin. It showed that vitronectin contains the entire 44-amino acid somatomedin B peptide at its NH2 terminus and, near its COOH terminus, a 34-amino acid glycosaminoglycan binding site in which half of the amino acids are basic residues. Three potential carbohydrate attachment sites are present in the sequence. An Arg-Gly-Asp sequence, which has previously been shown to be the cell attachment site in fibronectin, was found in vitronectin immediately after the NH2-terminal somatomedin B sequence. No other homologies with fibronectin were found. The Arg-Gly-Asp sequence appears to constitute the cell attachment site of vitronectin, since it is in the region where we have previously localized the cell attachment site, its presence correlate with cell attachment activity among the insert-coded polypeptides, and because previous results have shown that synthetic peptides containing the Arg-Gly-Asp sequence inhibit the cell attachment function of vitronectin. The discovery of an Arg-Gly-Asp cell attachment site in a protein with a known cell attachment function emphasizes the general importance of this sequence in cell recognition.     

Nucleotide sequence of the operators of lambda ultravirulent mutants.

The nucleotide sequence of the operators of ultravirulent mutants of lambda, able to grow on host cells with elevated repressor levels, was determined. It appears that ultravirulence in lambda requires multiple mutational events at the operator sequences. OL1, OL2, and OL3 operator sites are the target of mutational changes in ultravirulent phages indicating that these sites participate in vivo in repression of the PL promoter. No changes were found in the OR3 sequence, in contrast there is a mutation in OR2 and two mutations in OR1, in both lambda 668 and lambda 2668 phages. This mutated operator structure accounts for the constitutive expression of their PR promoter either in cells overproducing the lambda repressor or in cells overproducing the cro gene product. A model of the structure of the lambda operator site is proposed. The nucleotide sequence in each site can be divided into two functionally different subsets, one of which is recognized by the repressor while the other stabilizes the repressor-operator interaction.     

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.     

The structure of the trpE, trpD and 5' trpC genes of Bacillus pumilus

The nucleotide (nt) sequences of the Bacillus pumilus trpE, trpD and 5' portions of trpC genes have been determined. Genetic analysis suggested the presence of an internal promoter upstream from the trpC gene, yet no typical consensus sequences were found. The nt and amino acid sequence homologies between the B. pumilus, Bacillus subtilis and Escherichia coli trp genes are presented.     

The nucleotide recognized by the Escherichia coli A restriction and modification enzyme

The nucleotide recognition sequence for the restriction-modification enzyme of Escherichia coli A (EcoA) has been determined to be GAG-7N-GTCA. This sequence is fairly similar, but distinctly different from the two other type I restriction enzyme recognition sites known for E. coli B and E. coli K12, respectively. N6-adenosine methylation has been observed at nucleotide positions 2 and 12 within that sequence after modification by EcoA. As a reference point for mapping the single EcoA site in lambda, the position of lambda point mutation Oam29 has been determined also.     

Nucleotide sequence of the chi recombinational hot spot chi +D in bacteriophage lambda.

Chi sites in bacteriophage lambda stimulate recombination promoted by the RecBC pathway of Escherichia coli. Mutations which create these sites occur at four widely separated loci in lambda. We report here the nucleotide sequence surrounding the site of one of these loci, chi D, located near the S gene. The mutations creating the active Chi site, designated chi +D, are transversions from CG to AT. This mutation, like the chi +B and chi +C mutations previously analyzed, leads to a nucleotide sequence common to all three active chi sites.     

The extent of DNA sequence required for a functional bacterial attachment site of phage lambda.

We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration. A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency. We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo. We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.     

Molecular cloning of human prostate specific antigen cDNA

A lambda gt11 clone encoding prostate specific antigen has been isolated from a human prostate cDNA library. The cDNA insert of 1415 nucleotides hybridizes specifically to a prostate mRNA species of 1.5 kb. The nucleotide sequence codes for part of a signal peptide, a short propiece and the mature protein of 237 amino acid residues. The Mr for the non-glycosylated protein was 26,089. One potential site for N-linked carbohydrate attachment was identified. The primary structure shows extensive homology with proteases of the kallikrein family.     

Multiple origins of the human glycophorin Sta gene. Identification of hot spots for independent unequal homologous recombinations.

Human glycophorin Sta (HGpSta), one of the structural variants of erythrocyte membrane sialoglycoproteins, is encoded by a delta-alpha hybrid gene that arose from a single unequal crossover between the parent HGpB(delta) and HGpA(alpha) genes. We report here the identification of two new HGpSta genes (type A and type B) in four unrelated Sta heterozygotes from two ethnic groups. These Sta genes represent distinct genetic isoforms that differ from the previously reported Sta gene (type C) in the location of crossing-over sites. Comparison of nucleotide sequences among HGpB(delta), HGpA(alpha), and HGpSta type A genes revealed that the delta-alpha unequal crossover for the Sta type A gene occurred 110-246 base pairs downstream from pseudoexon III. In the crossing-over site of this Sta gene, an AT-rich sequence lying 3' to a nonameric palindrome was found to be highly similar to the lambda phage attachment site, att B, in inverted orientation. In the Sta type B gene, the delta-alpha crossing-over point was localized to an AG-rich sequence that is 302-490 base pairs downstream from pseudoexon III. Multiple lambda chi-like elements were identified at the crossover boundaries and within the breakpoint of this Sta gene. These results suggest strongly that recurrent and independent unequal recombination events have occurred in the formation of multiple Sta genes and that particular genomic sequences are important in defining the recombination sites for these homology-driven processes.     

DNA sequence at the end of the cI gene in bacteriophage lambda.

The nucleotide sequence of 57 base pairs near the end of the cI gene in phage lambda is presented. This sequence was determined by direct sequencing techniques and includes the codons for 11 carboxyterminal aminoacids of the cI product, the lambda repressor. The sequence reveals that the cI gene, which has recently been shown to have a unique initiation region, is terminated by a UGA codon. A GUG triplet, which could act as a translation start signal for the rex gene occurs 8 base pairs beyond the cI termination codon. This GUG triplet is preceded by a sequence that could serve as a strong ribosome binding site for the rex message.     

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.