recA基因通过同源重组途径参与由整合质粒发动的大肠杆菌染色体复制

我们在前文中报道由整合的F'质粒所发动的大肠杆菌染色体的复制依赖于recA基因。本文报道有关recA、recB、recC以及lexA等在染色体复制中的作用,实验结果说明,recA基因通过同源重组途径而不是通过SOS途径参与复制,而且recA基因和Chi热点无关。实验结果还说明,RecBC酶的依赖于ATP的双链DNA外切核酸酶活性和recA基因的作用无关。     

HAUL-OUT PATTERNS, SITE FIDELITY AND ACTIVITY BUDGETS OF MALE HOOKER'S SEA LIONS (PHOCARCTOS HOOKERI) ON THE NEW ZEALAND MAINLAND

Diurnal and seasonal haul-out patterns, site fidelity and activity budgets of individually identified Hooker's sea lions were studied for two years at Papanui Beach, Otago Peninsula, New Zealand.
Fourteen male sea lions were identified. Lengths ranged from 1.65 m–2.28 m and estimated ages from 2-11 yr. The population consisted of four sexually and socially mature (potentially breeding), eight sexually mature but socially immature and two immature males. Most haul-outs (95.6%) were by nine identifiable individuals (Residents) returning on a regular basis, suggesting a high degree of site specificity. Emigration and recruitment were low in relation to the length of the study. Daily arrivals (mean = 0844 h, SD = 1.49) and departures (mean = 1802 h, SD = 1.18) indicate nocturnal feeding. During March 1986 sea lions spent 43.8% of each day ashore (= 78% of daylight hours). Numbers of sea lions hauled out declined in both breeding seasons; in 1986/87 this was due to a decrease in haul-out frequency of resident animals. All but one of these sea lions returned after the breeding season.
Sea lions preferentially selected the middle and the extreme ends of the beach as haul-out sites. During winter use was made of the grass dunes as haul-out areas.
There were significant differences in the frequencies of behavioral activities between summer and winter; more time was allocated to resting in summer.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12

Summary Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1^intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1^intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1^intB indicated that this element was integrated at the same attP site as previously identified for SSV1^intA. A comparison of the DNA sequences at the left attachment sites of SSV1^intA and SSV1^intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 by inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed. Offprint requests to: W. Zillig     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

The photocycle and the structure of iron containing bacteriorhodopsin —a kinetic and Mössbauer spectroscopy investigation

Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe²⁺ or Fe³⁺ ions. ⁵⁷Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3⁺ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2⁺ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectroscopy show that the purple membrane becomes flexible only above 220 K. At the interface between membrane and bulk water the mobility is comparable to that of proteins with hydrophilic surfaces. The photocycle of Fe ³⁺-bR is slowed down compared to native bR. 3–5 Fe³⁺/bR are sufficient to inhibit the photocycle turnover by one order of magnitude. This specific effect is also found with Cr³⁺, though it is less pronounced. Mössbauer spectra of Fe³⁺-bR at 4 K reveal that iron nuclei are spin-coupled, indicating their close spatial proximity. It is proposed that iron trinuclear clusters interact with the proton uptake site of bR. Offprint requests to: M. Engelhard     

白细胞介素2受体α链基因转录起始点和5'调控区结合蛋白的初步研究

 借助于5'和3'末端删切后重建的IL-2R a链基因调控区次级克隆,在体外合成有放射性同位素参入的反意义RNA探针与总RNA进行液相杂交,结果表明TPA或PHA分别活化的T细胞在IL-2R a链表达过程中都在不同程度上有选择地利用了调控区内分别为-58(5')和+1(3')位两个转录起始点中3'转录起始点。热休克使PHA活化细胞更明显地利用+1位点。PHA诱导Jurkat细胞表达IL-2RamRNA斑点杂交证实,Jurkat细胞在活化16小时表达IL-2Ra基本达到高峰,至24小时已明显下降。根据这一规律提取PHA诱导活化15小时的Jurkat细胞S100和NE,进行有关结合蛋白的研究,初步结果显示磷酸纤维素柱的KCI洗脱组分中存在着DNA结合蛋白,有关结合蛋白性质的研究正在进行中。