Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. ODS C18 column (100 x 2.1 mm, Brownlee Laboratories) equilibrated with 0.08% trifluoroacetic acid/1% acetonitrile and were eluted with a linear gradient of 1-60% acetonitrile. Partially purified peptides were re-applied to the same column equilibrated with 50 mM ammonium acetate (pH 7.0) and were eluted with a linear gradient of 0-60% acetonitrile. Amino acid sequences of three peptides were determined using a Beckman ABI 477A peptide sequencer.Oligodeoxynucleotide Probes. Single long antisense oligonucleotide probes were designed according to Lathe (11) and synthesized on an Applied Biosystems model 380B DNA synthesizer. Oligonucleotides were 32P-labeled using T4 polynucleotide kinase.Isolation of cDNA Clones by Oligonucleotide Screening. A human Jurkat T-lymphoblast cDNA library in Agtll (obtained from H. Kataoka, Imperial Cancer Research Fund, Clare Hall Laboratories) was screened using 32P-labeled oligonucleotide probes. Hybridization was carried out at 37°C for 18 hr in 6x SSC (0.9 M NaCl/0.09 M sodium citrate, pH 7)/5 x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin)/0.1% SDS containing sheared, denatured herring sperm DNA (100 ,ug/ml). Filters were washed in 6x SSC/0.1% SDS for 20 min at both 50°C and 55°C.Isolation of cDNA Clones by Complementation in S. cerevisiae. The recipient yeast strain used in transformations was L94-4D (MATa cdc9-7 ura3 trpl), constructed by L.H.J., and its congenic wild-type parent was YNN27 (obtained from

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic k-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase-and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos-the cis-element required for linearization-was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.

Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.

The reproductive traits and the monthly larval abundance of the mantis shrimp Oratosquilla oratoria were investigated in Tokyo Bay, Japan, in 2002.