Capacity building of primary care physicians of the tea garden hospitals in Dibrugarh, Assam: A demonstration project.

BACKGROUND: The three most commonly occurring cancers in India are those of the breast, uterine cervix, and lip or oral cavity, together accounting for approximately 34% of all cancers. All the three cancers are amenable to prevention, early detection, and treatment through which the morbidity and mortality due to these cancers can be reduced. This pilot study was conducted to assess the operational feasibility of the national cancer screening guidelines. METHOD: This study was conducted in the Dibrugarh district of Assam in seven tea garden hospitals which serve as the primary health centers for the tea estate population in the Northeast region of India. The study intervention was a three-day training package designed to train primary care physicians in population-based screening for oral, breast, and cervical cancers. Knowledge evaluation and skill assessment were performed with a validated questionnaire and checklist, respectively. RESULTS: Pre and posttraining knowledge assessment showed significant gain in the knowledge levels of the participants in all topics. The greatest knowledge increase was seen in breast cancer (96.3%), followed by cervical cancer (57.5%), oral cancer (35.5%) and general cancer-related information (16.7%). The skill assessment done for each participant individually at the end of the training indicated a need for retraining all participants in breast cancer screening. CONCLUSION: The learnings from this study will be of great help in scaling up the capacity building programme for cancer screening when the nation-wide population-based cancer screening programme will be rolled out in the country.

Electro-Physiology of Coupling Model and Its Impact on Naja Kaouthia Venom Treated Sciatic Nerves of Toad.

Demyelination in peripheral nerves causes dysfunction of slowing down and stoppage of nerve impulses causing many neurological diseases, such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, etc. This paper aims to develop a recovery model having interaction of a demyelinated nerve with a normal myelinated nerve. We validated the model by coupling between peripheral nerve of toad (demyelinated with Naja kaouthia venom) and a normal nerve of toad. An increase in both nerve conduction velocity as well as compound action potential amplitude is observed in the repetition of the experiments indicating gradual recovery of the patients. The significance behind this work is to suppress the malfunctioning of the demyelinated nerve by the normal electro-physiological activity of the normal nerve for speedy recovery using coupling model. The recovery model will be used in the treatment of neurological disorders with the influence of normal neuro physiological properties of a normal adjacent nerve.

Quantifying Demyelination in NK venom treated nerve using its electric circuit model.

Reduction of myelin in peripheral nerve causes critical demyelinating diseases such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, etc. Clinical monitoring of these diseases requires rapid and non-invasive quantification of demyelination. Here we have developed formulation of nerve conduction velocity (NCV) in terms of demyelination considering electric circuit model of a nerve having bundle of axons for its quantification from NCV measurements. This approach has been validated and demonstrated with toad nerve model treated with crude Naja kaouthia (NK) venom and also shows the effect of Phospholipase A2 and three finger neurotoxin from NK-venom on peripheral nerve. This opens future scope for non-invasive clinical measurement of demyelination.

Evidence for pre- and post-power stroke of cross-bridges of contracting skeletal myofibrils.

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10(-15) L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.

Risk of hepatitis C infection among injection drug users in Mizoram, India.

BACKGROUND & OBJECTIVE: Prevalence of injection drug users (IDUs) is high in the northeastern region of India. This coupled with unsafe injecting practices as well as practice of tattooing in remote tribal areas call for baseline data on the prevalence of parentally transmitted viral diseases. In the present study we aimed to measure the risk behaviours and seroprevalence of hepatitis C virus (HCV) antibodies amongst IDUs of Mizoram, a State of the northeast India. METHODS: A cross-sectional study was conducted in 2004-2005 amongst IDUs (including female sex workers) who had injected in the past six months and were unaware of their HCV/HIV status. They were recruited from various drop-in centers from Aizawl, Mizoram, and screened for anti-HCV antibodies using 3(rd) generation HCV EIA and recombinant immunoblot assay (RIBA). RESULTS: The prevalence of HCV antibodies was 71.2 per cent among the active IDUs. On univariate analysis increasing duration of injection, syringe sharing and heroin (diacetylmorphine) injectors were at a significantly higher risk of acquiring HCV antibodies (P<0.001). On multivariate analysis, HCV antibody prevalence showed a strong association with the type of drugs injected (P=0.001), frequency of injecting (P=0.013), multiplicity of drugs abused (P=0.004), and needle syringe sharing (P=0.003). INTERPRETATION & CONCLUSION: Unsafe injecting practices were found to be associated with a higher risk of acquiring hepatitis C infection. Our findings showed that syringe and needle exchange programme alone was not sufficient as a preventive strategy for control of hepatitis C infection among IDUs of Aizawl.

Arsenic concentrations in rice, vegetables, and fish in Bangladesh: a preliminary study.

Arsenic contaminating groundwater in Bangladesh is one of the largest environmental health hazards in the world. Because of the potential risk to human health through consumption of agricultural produce grown in fields irrigated with arsenic contaminated water, we have determined the level of contamination in 100 samples of crop, vegetables and fresh water fish collected from three different regions in Bangladesh. Arsenic concentrations were determined by hydride generation atomic absorption spectrophotometry. All 11 samples of water and 18 samples of soil exceeded the expected limits of arsenic. No samples of rice grain (Oryza sativa L.) had arsenic concentrations more than the recommended limit of 1.0 mg/kg. However, rice plants, especially the roots had a significantly higher concentration of arsenic (2.4 mg/kg) compared to stem (0.73 mg/kg) and rice grains (0.14 mg/kg). Arsenic contents of vegetables varied; those exceeding the food safety limits included Kachu sak (Colocasia antiquorum) (0.09-3.99 mg/kg, n=9), potatoes (Solanum tuberisum) (0.07-1.36 mg/kg, n=5), and Kalmi sak (Ipomoea reptoms) (0.1-1.53 mg/kg, n=6). Lata fish (Ophicephalus punctatus) did not contain unacceptable levels of arsenic. These results indicate that arsenic contaminates some food items in Bangladesh. Further studies with larger samples are needed to demonstrate the extent of arsenic contamination of food in Bangladesh.

HCV activity in an isolated community in north east India.

Prevalence of anti-HCV among the people at risk and general population were reported across the globe. We investigated HCV activity among the members of "Lisu" community settled in a remote and isolated area of Changlang District, Arunachal Pradesh during 1999-2000. The families were scattered with 380 households. Blood samples were collected from 76 (35 males and 45 females) apparently healthy individuals from randomly selected 10% families. Sera were processed for detection for antibody to HCV by using 3rd generation ELISA kit. All the persons were within the age of 18-98 years and 75% of them were uneducated and 92% were cultivators. The prevalence of anti-HCV was found to be very high (7.89%). Since the HCV activity is high in an isolated community, transmission dynamics study will be interesting for this epidemiologically important viral disease.

Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene.

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from -35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (-35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the -10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the -10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.

Risk factors for cancer nasopharynx: a case-control study from Nagaland, India.

BACKGROUND: A high incidence of nasopharyngeal carcinoma has been reported from Nagaland, though it is considered to be a rare neoplasm in India. No case-control study to identify the risk factors of cancer nasopharynx has been conducted in this region. This study was undertaken to identify dietary and environmental risk factors for nasopharyngeal carcinoma relevant to this region. METHODS: A matched case-control study using neighbourhood controls was conducted. For each of the 47 cases identified, 2 apparently healthy neighbourhood controls were matched for age, sex and ethnicity. All information on dietary, environmental, social and demographic factors was collected. Univariate and multivariate logistic regression analysis using maximum likelihood method was used to analyse data. RESULTS: Consumption of smoked meat was found to be the risk factor for nasopharyngeal carcinoma (adjusted odds ratio = 10.8; 95% CI 3.0-39.0). History of using herbal nasal medicine was also found to be associated with nasopharyngeal carcinoma (OR = 21.9, CI = 6.8-71.4). However, exposure to a smoky atmosphere, betel-nut chewing, use of smokeless tobacco products, smoking and drinking habits were not found to be associated with nasopharyngeal carcinoma. CONCLUSION: This study reveals an association of nasopharyngeal carcinoma with consumption of smoked meat in Nagaland. The use of herbal nasal medicine seems to be an additional risk factor for nasopharyngeal carcinoma in Nagaland and needs further assessment.

The replication origin of Azotobacter vinelandii.

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.

Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I.

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. A negative cis-acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.

A- and T-tract-mediated intrinsic curvature in native DNA between the binding site of the upstream activator NtrC and the nifLA promoter of Klebsiella pneumoniae facilitates transcription.

The nif promoters of Klebsiella pneumoniae must be activated by proteins bound to upstream sequences which are thought to interact with the sigma54-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the nifLA promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the Escherichia coli lacZ gene of the DNA fragment containing the nifLA promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in K. pneumoniae and E. coli was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the nifLA promoter of K. pneumoniae.

An upstream element containing an ETS binding site is crucial for transcription of the human presenilin-1 gene.

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the most active fragment from -118 to +178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatoma HepG2 cells. 5' deletions revealed that a crucial element controlling over 90% of the promoter activity in these cell lines is located between -22 and -6. A mutation altering only two nucleotides of the ETS consensus sequence present at -12 (GGAA to TTAA) has a similar effect. Electrophoretic mobility shift assays showed that a set of specific complexes between nuclear factors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experiments in DNase I footprinting correlated with electrophoretic mobility shift assays and showed that only one of several footprints over the PS1 promoter is eliminated by competition with an ETS consensus oligonucleotide. It extends from -14 to -6 and surrounds the ETS motif present at -12. Thus, a crucial ETS element is present at -12 and binds a protein(s) recognizing specifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reactivity with Ets-1 and Ets-2 proteins, thus confirming that an ETS transcription factor(s) recognizes the -12 motif. Several Sp1 binding motifs at positions -70, -55, and +20 surround this ETS element. Competition DNase I footprinting showed that Sp1-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5' and 3' deletions indicated the presence of positive promoter elements between -96 and -35 as well as between +6 and +42. Thus, transfection and footprinting assays correlate to suggest that Sp1 transcription factor(s) bind at several sites upstream and downstream from the initiation site and activate the transcription of the PS1 promoter. Sequences downstream from the transcription initiation site also contain major control elements. 3' deletions from +178 to +107 decreased promoter activity by 80%. However, further deletion to +42 increased promoter activity by 3-4-fold. Collectively, these data indicate that sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protein-protein interactions between factors recognizing downstream and upstream sequences are involved.

A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells.

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the -128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans-acting factors that interact with cis-acting elements present in the first non-translated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; -84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans-acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to -85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous from of an autosomal-dominant disorder.

Analysis of upstream activation of the vnfH promoter of Azotobacter vinelandii.

BAL-31 deletion products of the DNA fragment containing the vnfH promoter and upstream region, when cloned in a transcriptional fusion vector and analyzed for vnfH expression in Azotobacter vinelandii, revealed that the upstream activator sequence of the vnfH promoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of the vnfH promoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of the vnfH promoter, respectively, are both required for vnfH expression. Addition of four nucleotides in the intervening sequence between the vnfH promoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from the vnfH promoter in Azotobacter vinelandii, whereas addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression from vnfH promoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum.

A spontaneous mutant derivative of Azotobacter vinelandii CA12 (delta nif HDK), which vanadium-dependent nitrogen fixation is not inhibited by molybdenum (A. vinelandii CARR), grows profusely on BNF-agar containing 1 microM Na2MoO4, alone or supplemented with 1 microM V2O5. The expression of A. vinelandii vnfH::lacZ and vnfA::lacZ fusions in A. vinelandii CARR was not inhibited by 1 mM Na2MoO4, whereas molybdenum at much lower concentration inhibited the expression of vnfH::lacZ and vnfA::lacZ fusions in A. vinlandii CA12. The mutant also exhibited normal acetylene reduction activity in the presence of 1 microM Na2MoO4. The expression of A. vinelandii nifH::lacZ fusion in A. vinelandii CARR was low even though the cells were cultured under non-repressing conditions with urea as nitrogen source in the presence of Na2MoO4. The molybdenum content of A. vinelandii CARR cells was found to be about one-fourth that of A. vinelandii CA12. No nitrate reductase activity could be detected in A. vinelandii CARR when the cells were cultured in the presence of 10 microM Na2MoO4, whereas A. vinelandii CA12 exhibited some activity even with 100 pM Na2MoO4.

Bancroftian filariasis in Namrup tea estate, district Dibrugarh, Assam.

Filariasis survey in a randomly selected tea estate of district Dibrugrah revealed 6.7% infection of Wuchereria bancrofti in labour population with microfilaria (mf) rate of 7.6% in males and 5.9% in females. The mf rate increased progressively with the age which however, dropped in 31-40 age group of males and in 41-50 age group of females. Chronic filariasis diseases rate was 2.7%. The involvement of genitals in manifesting chronic filariasis was significantly higher than of the lower extremities. Infection and infectivity rates in the vector mosquito, Culex quinquefasciatus were 6.1% and 4.6% respectively with mean L3 load per infective mosquito of 8.5. Drains, land, peridomestic ditches were chief breeding habitats of Cules quinquefasciatus in the tea estate.

Genes implicated in the pathogenesis of Alzheimer's disease.

Both the early and late-onset Alzheimer's disease affect millions of people throughout the world. A number of molecules have been implicated in the pathogenesis of Alzheimer's disease. These include presenilin 1 and 2 (PS1 and PS2), a beta-amyloid peptide, and tau protein. Presenilin 1 and 2 genes implicated in the early-onset familial Alzheimer's disease have been cloned. Both PS1 and PS2 are integral membrane proteins and may function as receptors or channel proteins. Missense mutations in PS1 and PS2 genes have been found in families that cosegregate with early-onset Alzheimer's disease. Overexpression of the mutated PS1 gene produced amyloid plaques in the brain of transgenic mice. Secreted beta-amyloid protein similar to that in the senile plaques of Alzheimer's disease was found to be elevated in fibroblast media from subjects with PS1 or PS2 mutations. Transgenic mice which carried the mutant form of the beta-amyloid precursor protein gene expressed high concentrations of mutant copy of the gene and exhibited abundant amyloid plaques in the brain and memory loss. The mutated PS2 gene enhanced apoptotic activity. This enhanced apoptotic activity may accelerate the process of neurodegeneration leading to an earlier age in the onset of the disease. Identification of lesions in the molecules that are important in the Alzheimer's disease should allow developing therapeutic approaches for its treatment.

Mercury speciation and accumulation in Bangladesh freshwater and anadromous fish.

Total mercury concentration in the muscle of 417 fish of 12 common freshwater and four anadromous species from Bangladesh were low, varying from 2 to 430 ng/g fresh wt. Depending on Hg speciation, three types of accumulation mechanisms were defined. Type I covers the majority of species and describes a pattern widely accepted as 'normal', with increasing levels of organic (methyl) mercury with length (age), combined to a low and constant inorganic level. This accumulation pattern leads to a relative increase of the organic mercury fraction with age, eventually reaching 90-100% of organic mercury in full grown specimens. Type II is found in both planktivorous genera only and showed increasing levels of inorganic mercury combined to low and constant organic mercury levels, leading to a relative decrease in organic mercury fraction with age. This unexpected pattern was only reported in cases of some marine species where it seemed to be linked to demethylation mechanisms or regional influences on Hg levels. A third intermediate accumulation pattern with increasing concentrations of both the organic and the inorganic Hg fraction with age was found in one bottom dwelling species only. The implications of these observations for the accumulation mechanisms of mercury in fish are discussed.

Characterization and mutagenesis of the leucine biosynthetic genes of Azotobacter vinelandii: an analysis of the rarity of amino acid auxotrophs.

An 8.7 kb region of the Azotobacter vinelandii chromosome has been analyzed by genetic complementation and nucleotide sequencing. The sequence reveals that leuC and leuD comprise an operon- and leuB is adjacent to leuD. leuA was not detected. Experiments involving lac fusion constructs have confirmed the existence of separate promoters for leuC-leuD and for leuB. Primer extension studies have localized the transcription initiation sites of the leuC-leuD operon and also of the leuB operon. Five more open reading frames showing homology with the Escherichia coli genes yoh1, ibpB, cynR, asd and usg1 have also been found. Auxotrophic mutations are rare in A. vinelandii. We have been able to generate, for the first time, stable mutations in leuB, leuC and leuD by insertion of various gene blocks in vitro and integration by double crossover in vivo. Homogenotization of the mutation into all of the multiple chromosomes of A. vinelandii has been achieved. Evidence has been obtained suggesting the presence of a permease in A. vinelandii capable of leucine transport. Possible reasons for the dearth of auxotrophic mutations in A. vinelandii are discussed.