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N Hasan

Publications and source records attributed to N Hasan.

52 records · Page 3Linked to original sources
PubMed1986

A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme.

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Deletion↗
PubMed1986

Boundaries of the nutL antiterminator of coliphage lambda and effects of mutations in the spacer region between boxA and boxB.

To study the relationship of sequence to function for the phage lambda nutL transcriptional antiterminator, we cloned the 354-bp HincII lambda DNA fragment (coordinates 35261-35615; Daniels et al., in Lambda II, 1983, pp. 485-486 and 618-619) between the plac promoter and Rho-dependent or Rho-independent terminators (t) in the plac-t-galK plasmid derived from vector pKO3, and assayed the galK expression in Escherichia coli hosts in the presence or absence of the N gene product. The 354-bp fragment displayed the complete antitermination activity, as did several shorter fragments obtained by restriction cutting, exonucleolytic deletions and ligations. The minimal length of cloned and fully active nutL comprises 43 bp and consists (in the 5'-to-3' order) of a 10-bp sequence upstream from boxA, the 8-bp boxA (5'-CGCTCTTA-3'), the 7-bp A-B spacer between boxA and boxB; the 17-bp boxB (nutL core; 5'-AGCCCTGAAGAAGGGCA-3') and 1-bp downstream from the nutL core. Deletion of the 10-bp sequence upstream from boxA reduces anti-termination by 40%. Deletion of both that sequence and boxA reduces antitermination by 90% at both 30 degrees C and 42 degrees C. Most of the deletions entering boxB abolish antitermination. Also, some of the small internal deletions within the 7-bp A-B spacer region have a strong negative effect on the nutL function, when transcription is from the plac promoter.

Bacteriophage lambda↗
PubMed1986

Effect of the promoter structure on the nutL transcription antitermination function.

Several boxA-less 74-bp nutL antiterminator fragments do not function as antiterminators of plac-promoted transcription, but are active with the pp and p'R promoters at 30 degrees C. At elevated temperatures (42 degrees C), these defective 74-bp nutL modules retain 50% of their activity when controlled by the p'R promoter; with the pp promoter the antitermination activity is 5-10 times less efficient. Similarly, deletions of 1-3 bp in the spacer region between the boxA and boxB subunits of nutL (which abolish antitermination promoted by plac), allow rather efficient but thermosensitive antitermination when controlled by the pp or p'R promoter. These results point to possible conditional interactions between the promoter and antiterminator elements and functions. Also noted were unexpectedly slow mobilities of the p'R-nutL-bearing fragments, suggesting some unusual secondary structures.

Bacteriophage lambda↗
PubMed1985

Thermosensitivity of a DNA recognition site: activity of a truncated nutL antiterminator of coliphage lambda.

Antitermination is an important transcriptional control. In bacteriophage lambda, the presence of the nut antiterminators between the promoters and terminators results in relatively unhindered transcription when the lambda N gene product and necessary host factors are supplied. This antitermination system has been rendered thermosensitivity by modification of the nut site. A fragment of lambda DNA [74 base pairs (bp) in length]that contained the 17-bp nutL core sequence, but lacked the 8-bp boxA sequence, was cloned in a pp-N-tL1-galK plasmid between the pp promoter and gene N. This fragment mediated antitermination of transcription at 30 degrees C, as measured by assaying galK gene expression in Escherichia coli. At 42 degrees C, however, antitermination at the lambda tL1 terminator was abolished. Antitermination at 42 degrees C was restored by replacing the 74-bp nutL fragment with longer sequences containing both nutL and boxA or by cloning a synthetic boxA sequence ahead of the 74-bp nutL fragment. Thus, efficient antitermination required both boxA and the 17-bp nutL core, with the latter becoming conditionally defective when the boxA sequence was deleted.

Bacteriophage lambda↗
PubMed1985

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module.

We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1978

Purification and characterization of NADPH-dependent flavin reductase. An enzyme required for the activation of chorismate synthase in Bacillus subtilis.

NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.

Bacillus subtilis↗
PubMed1978

Purification and properties of chorismate synthase from Bacillus subtilis.

Chorismatic synthase was purified to apparent homogeneity from Bacillus subtilis. The enzyme required NADPH-dependent flavin reductase, Mg2+, NADPH, and flavin (FMN or FAD) for activity. The molecular weight of chorismate synthase was 24,000 as determined by sodium dedecyl sulfate (SDS)-gel electrophoresis. The enzyme was also isolated in a complex form associated with NADPH-dependent flavin reductase and another enzyme of the aromatic amino acid pathway, dehydroquinate synthase. On SDS-gel electrophoresis, this form was resolved into three bands with molecular weights of 13,000, 17,000, and 24,000. The enzyme complex was easily dissociated and the dissociation resulted in a change in the chromatographic properties of NADPH-dependent flavin reductase which was no longer retained on phosphocellulose whereas chorismate synthase was still adsorbed. Chorismate synthase activity was linear with time and protein concentration, whereas partially purified preparations showed a significant lag period before the reaction took place. Moreover, crude or partially purified enzyme preparations were completely inactivated by dilution and the activity could be recovered by addition of flavin reductase. A possible role of NADPH-dependent flavin reductase in the activation and regulation of chorismate synthase activity is discussed.

Bacillus subtilis↗
PubMed1978

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.

Bacillus subtilis↗
PubMed1974

Induction of beta-galactosidase in Lactobacillus plantarum.

beta-galactosidase (beta-galactoside galactohydrolase, EC 3.2.1.23) is inducible in Lactobacillus plantarum by d-galactose or thiomethyl galactoside, and to a much lesser extent by lactose, isopropyl thiomethyl galactoside, and d-fucose. Isopropyl thiomethyl galactoside is a competitive inhibitor of the enzyme with a K(i) of 4.2 mM. The K(m) of the crude enzyme for o-nitrophenyl beta-d-galactoside is 0.87 mM. Induction also requires a source of energy and amino acids. Chloramphenicol and actinomycin D inhibited induction. d-Glucose, d-fructose and to a lesser extent maltose and d-mannitol inhibited enzyme synthesis. Methyl-alpha-d-glucopyranoside was not inhibitory. Glucose exerts its effect through its ability to exclude galactose or lactose entry into the cell. The uptake of lactose and the metabolism of galactose by preinduced cells is severely inhibited by glucose. But neither galactose nor lactose severely affected the uptake of glucose by preinduced cells. Thus, glucose acts through catabolite inhibition, i.e., transport of inducer rather than repression through transcription or related mechanisms. This is supported by the inability of cyclic nucleotides to relieve the inhibition produced by glucose or to stimulate induction. Furthermore, intracellularly produced glucose did not inhibit enzyme synthesis. Acetate and mevalonate, the precursors of membrane lipids, stimulate induction independently of their effect on growth. Homobiotin partially abolished the acetate effect but did not inhibit induction or growth.

Acetates↗
PubMedDate not supplied

Client satisfaction with the nutrition education component of the California WIC program.

Past evaluation research has documented improved nutritional outcomes resulting from participation in the Special Supplemental Nutrition Program for Women, Infants and Children (WIC). However, these evaluations have not examined the program from the clients' perspective, nor have they examined the independent effect of the nutrition education component. The purpose of this study was to quantitatively and qualitatively examine client satisfaction with the nutrition education component of the California WIC program. The methodology consisted of two phases. During phase I of the study (the quantitative component), participants completed Client Satisfaction Surveys immediately following attendance of one nutrition class. During phase II (the qualitative component), four focus groups were conducted. All subjects were participants in the California WIC program. Client Satisfaction Surveys were completed by 2138 participants, and the focus groups included 29 participants. Results from both phases of the study indicated that client satisfaction with the nutrition education component of the California WIC program was high. Between 80% and 95% of participants responded positively to five satisfaction questions, and focus group participants unanimously agreed that the nutrition education was an essential component of the program. Hispanic participants were more likely than non-Hispanic Caucasians, Asians, or African Americans to respond positively to three of the five satisfaction questions. For two of the questions, the frequency of positive responses increased as age increased and decreased as education level increased. A small segment of clients reported some dissatisfaction by responding negatively to one or more of the satisfaction questions (4% to 20% of respondents). Some suggestions for improvement were made by survey respondents. Identification of some WIC participants who are not completely satisfied with the nutrition education that they have received, paired with differences in satisfaction across demographic variables, suggests the need for a personalized approach to WIC nutrition education.

Adolescent↗