Strand specificity of DNA unwinding by RecBCD enzyme.

RecBCD enzyme (exonuclease V) of Escherichia coli unwinds DNA, frequently forming asymmetric structures with two single-stranded tails of unequal length abutting a single-stranded loop at the junction with double-stranded DNA. Their lengths are consistent with the longer tail being one strand of the duplex and the loop plus the shorter tail being the other strand. The strand polarity of the unwinding was determined by labeling the 3' or 5' ends of duplex DNA with biotinylated nucleotides, reacting the DNA with RecBCD enzyme, and distinguishing the labeled ends, in the electron microscope, by their binding to streptavidin-gold complex. The shorter tail was formed from the DNA strand with its 3' terminus at the duplex end where RecBCD enzyme entered. We conclude that RecBCD enzyme unwinds DNA by forming a loop on the strand with a 3' end at the entry point. This result is concordant with a previously proposed model of recombination, which we discuss.

Localization of an ataxia-telangiectasia gene to chromosome 11q22-23.

Ataxia-telangiectasia (AT) is a human autosomal recessive disorder of childhood characterized by: (1) progressive cerebellar ataxia with degeneration of Purkinje cells; (2) hypersensitivity of fibroblasts and lymphocytes to ionizing radiation; (3) a 61-fold and 184-fold increased cancer incidence in white and black patients, respectively; (4) non-random chromosomal rearrangements in lymphocytes; (5) thymic hypoplasia with cellular and humoral (IgA and IgG2) immunodeficiencies; (6) elevated serum level of alphafetoprotein; (7) premature ageing; and (8) endocrine disorders, such as insulin-resistant diabetes mellitus. A DNA processing or repair protein is the suspected common denominator in this pathology. Heterozygotes are generally healthy; however, the sensitivity of their cultured cells to ionizing radiation is intermediate between normal individuals and that of affected homozygotes. Furthermore, heterozygous females are at an increased risk of breast cancer. These findings, when coupled with an estimated carrier frequency of 0.5-5.0%, suggest that (1) as many as one in five women with breast cancer may carry the AT gene and that (2) the increased radiation sensitivity of AT heterozygotes may be causing radiation therapists to reduce the doses of radiation used for treating cancer in all patients. To identify the genetic defect responsible for this multifaceted disorder, and to provide effective carrier detection, we performed a genetic linkage analysis of 31 families with AT-affected members. This has allowed us to localize a gene for AT to chromosomal region 11q22-23.

Different reading frames are responsible for IS1-dependent deletions and recombination.

Two classes of plasmids in addition to the parent become apparent when plasmids that contain direct repeats of IS1. One class of plasmids has deleted sequences from the end of IS1 to nonrandom sites within the plasmid. The appearance of these plasmids in the population requires intact insA and insB reading frames, but not insC. The other class of plasmids has undergone an exchange within the direct repeats of IS1 on the plasmid. Their appearance requires InsC but neither InsA nor InsB. The two reactions may represent two distinguishable steps in IS1 transposition. The InsC-catalyzed exchange is independent of RecA and resembles hologous recombination since the frequency of recombinants arising from exchanges in different regions of IS1 appears to be roughly proportional to the size of the region. InsC can also catalyze an exchange between direct repeats of non-IS1 DNA.

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs.

Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.

Recombination in recA cells between direct repeats of insertion element IS1.

The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the lambda pL promoter on the plasmid. A plasmid that contains mutant IS1 elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1. I discuss a possible role for the lambda pL promoter.

Role of the rel gene product in the control of cyclic adenosine 3',5'-monophosphate accumulation.

The presence of a relA mutant allele affects the kinetics of cyclic adenosine 3',5'-monophosphate accumulation during downshift from glucose to succinate. The nucleotide accumulates at the normal rate early in the downshift transition but continues to accumulate for a longer time in the relA mutant, leading to a two- to threefold excess by the end of the diauxic lag. Evidence is presented that this effect occurs independently of the accumulation of ppGpp.