von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.     

Nuclear activity from F9 embryonal carcinoma cells binding specifically to the enhancers of wild-type polyoma virus and PyEC mutant DNAs.

Although wild-type polyoma virus does not productively infect murine embryonal carcinoma (EC) cells, a number of mutants (PyEC mutants) that do infect undifferentiated EC cells have been isolated. All PyEC mutants have DNA sequence alterations within the enhancer region of the viral genome. This report describes an activity present in nuclear extracts of F9 EC cells which, by "footprint" analyses, binds specifically to a small region of about 20 base pairs (nucleotides 5180-5200) within the subregion of the polyoma enhancer designated as the B or beta element. While no difference in binding of factor was detected between wild-type polyoma enhancer and the enhancers of the PyEC mutants, PyF111 and PyF441, which had been selected for productive infection of F9 cells, definite differences between wild-type and mutants were observed in the digestion patterns of their naked DNAs with either DNAase I or exonuclease III. This difference was restricted to the region around the point mutation (nucleotide 5258) common to these mutant DNAs.     

Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice

Twelve Japanese rice cultivars were converted to CMS by asymmetric protoplast fusion with MTC-5A, the cytoplasm of which was derived from an indica rice, Chinsurah Boro II. With the exception of the cybrids that had a nucleus from Hoshiyutaka, most of these cybrid plants were sterile. The unique sequence downstream from the mitochondrial atp6 of MTC-5A was specifically amplified in the sterile cybrid plants by PCR. All progenies of the cybrid plants carrying this unique sequence were sterile. On the other hand, in some of the sterile cybrid plants in which the unique sequence was not amplified by PCR, fertility was recovered in their progenies. Somaclonal mutation may have caused sterility in these cybrids. Only the cybrid plants that had the unique sequence detected by PCR were CMS. Thus, the CMS plants can be selected rapidly and easily by PCR, at an early stage of plant regeneration. Soon after transplanting the regenerated plants to a green house, fertile cybrids and sterile cybrids produced by somaclonal mutation can be removed. These findings also show that the unique region downstream from atp6 is tightly linked with the CMS phenotype.     

Mechanism of T5-induced DNA polymerase. II. Characterization of the dead-end complex.

We have shown that bacteriophage T5-induced DNA polymerase replicates short primer-templates (400 to 600 nucleotides long) at a rapid rate initially, followed by a slower rate sustained for much longer periods (Das, S. K., and Fujimura, R. K. (1977) J. Biol. Chem. 252, 8700-8707). In order to explain the slower steady rate and the results of polymer-challenge experiments, we conjectured the presence of a "dead-end complex" formed by the enzyme with the primer-template at the end of the primer elongation process. In this communication we present evidence which indicates that the presumed complex shows a first order kinetics of decay with a half-life of 3.5 min at 37 degrees. Energies of activation for the steady phase of synthesis and the decay of the dead-end complex were both found to be about 23 kcal/mol. This indicates that the dissociation of the aforesaid complex might be the rate-limiting step during the steady phase of synthesis. Correlation between the salt-induced reduction in the half-life of the complex and the increase in the steady rate of synthesis is in agreement with the above mentioned possibility.     

Mechanism of T5-induced DNA polymerase. I. Replication of short primer templates.

Bacteriophage T5-induced DNA polymerase shows an initial phase of rapid synthesis, followed by a slower steady rate for much longer periods, with short DNA primer-templates (400 to 600 nucleotides long), in vitro. On extrapolating the line of steady rate back to 0 min, an intercept is obtained on the ordinate. With large DNA primer-templates, such as denatured T5 DNA (average chain length approximately 50,000 bases), the rate of synthesis remains constant and is equal to the initial rate obtained with short primer-templates. The zero time intercept was proportional to the amount of enzyme used and independent of temperature. Polymer challenge experiments indicate that the initial phase of rapid synthesis can be attributed to the processive mode of synthesis by T5 DNA polymerase. After synthesizing a stretch of DNA processively for about 200 nucleotide residues, the enzyme apparently forms a "dead-end complex" with the primer-templates used and must dissociate from the primer-template in order to resume synthesis. The average size of the product made processively, during various phase of synthesis, remains invariant and is in good agreement with the size of the zero time intercept per enzyme molecule.     

以化学纯饲料饲养北京的桃蚜

应用修改后的Dadd和Mitter(1966)全纯饲料配方配制成人工饲料饲养定居在北京温室烟草上的桃蚜 Myzus persicae可完成生活史井连续饲养3代。本文描述饲料配制、饲养和取食量测定的方法。这3代初羽化无翅孤雌胎生雌蚜的平均体重分别为:440±90.7μg,264±104.9μg和312±127.9μg。用放射性同位素稀释法测定取食量的结果得悉若虫期的总取食量每蚜约为1.74μg,相当于1.16μl。     

Information, discrimination and divergence in cytology. III. Optimization of classification of Papanicolaou smears

The performance of a cytology laboratory can be objectively quantitated as the total discrimination, a defined quantity of information. The total discrimination is dependent on the number of categories used in gynecologic cytology and on the corresponding histologic states; over-classification results in a higher rate of misinformation and reduced total discrimination. Total divergence is another measure of the association between cytologic categories and histologic states; in contrast to the total discrimination, the total divergence does not require a one-to-one correspondence between the cytologic categories and the histologic states. Using data from the Gynecologic Cytology Laboratory of the University of Minnesota, the total discrimination was maximized when gynecologic cytology used three categories of diagnosis, consisting of (1) normal, atypical benign or reactive atypia, (2) cervical intraepithelial neoplasia (CIN) and (3) all malignancies. The use of four categories, (1) normal, atypical benign or reactive atypia, (2) mild or moderate dysplasia, (3) severe dysplasia or squamous carcinoma in situ and (4) all malignancies, was almost equally informative. Observations on the total divergences resulted in similar conclusions. These findings generally support the recommendation of the consensus workshop sponsored by the National Cancer Institute (the Bethesda System nomenclature) to group all degrees of CIN into two large categories.     

基因体外诱变的一种新方法

在PCR的过程中,采用5溴脱氧尿苷三磷酸（BrdUTP）部分取代脱氧胸苷三磷酸(dTTP)的方法,对克隆的野油菜黄单胞菌的α淀粉酶基因进行了体外诱变。结果表明,BrdUTP浓度越高,诱变越强;浓度越低,诱变越弱。当BrdUTP浓度为dTTP的0.1%时,可以得到最多的正诱变结果。用LBSP鉴别培养基初筛,然后用Yoo改良法测定酶活,仅一轮诱变就获得了其表达产物α-淀粉酶的酶活分别降低了5倍和提高了20倍的两个突变基因。再以后者为PCR模板进行第二轮诱变,从而筛选到了α-淀粉酶的酶活提高40倍的突变体。此诱变方法克服了用碱基类似物在体内诱变由于核酸复制酶等的校正作用而造成诱变无效的难题,并为基因的体外诱变找到了一条新途径。