The Georgia Information Network for Public Health Officials (INPHO): a demonstration of the CDC INPHO concept.

The Georgia Division of Public Health is serving as the first demonstration model of a statewide implementation of the Centers for Disease Control and Prevention Information Network for Public Health Officials. The goals of the project are to make communication easy, to make information accessible, and to make data exchange as swift and as smooth as technology will allow. By having a state-of-the-art telecommunications network, public health officials will be able to rapidly collect health data and transform them into meaningful health status information that can be used to inform health policy decisions. The Georgia Information Network for Public Health Officials is a unique consortium of private and public sector partners funded with a grant from the Robert W. Woodruff Foundation. The project will install the telecommunications infrastructure and the supporting software to position Georgia public health as the health information leader of the state.

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia.

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO2 and is dispensable in the presence of high CO2 concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO2-binding activity.

Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII).

We isolated the ntrC gene from Bradyrhizobium japonicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked glnII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnII in both nitrogen-starved and nitrogen-excess cultures. Both mutants expressed glnII under oxygen-limited culture conditions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties.

This paper describes the construction of 18 cloned bacteriophage T7 late promoters with single point mutations. In vitro transcription experiments were used to characterize the properties of these promoters. Since the mutated promoters are cloned into identical backgrounds, differences seen in the transcription assays are directly attributable to the point mutations. All of the mutated promoters are less active than wildtype, but they can be divided into two types. Type A mutations map from -4 to +1 and reduce promoter activity when the template is linearized or when 60mM NaCl is added to the reaction buffer. Type B mutations map from -9 to -7 and reduce promoter activity under all conditions tested. At several sites all three possible point mutations are available. At these sites we observed hierarchies of base pair preference, as determined by promoter activity, that may indicate that T7 RNA polymerase interacts with groups in the major groove.

Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II).

The interactions of T7 RNA polymerase with T7 late promoters were studied by using quantitative footprinting with methidiumpropyl-EDTA X Fe(II) [MPE-Fe(II)] as the DNA cleaving agent. Class II and class III T7 promoters have a highly conserved 23 base pair sequence from -17 to +6. Among class III promoters the -22 to -18 region is also highly conserved. For a class II promoter, T7 RNA polymerase protects the -17 to -4 region from MPE-Fe(II) cleavage; when GTP is present, protection extends from -17 to +5 (noncoding strand). For a class III promoter, protection extends from -20 to -4 and in the presence of GTP from -20 to +5 (noncoding strand). The protected regions for the coding strands of both promoters were nearly identical with that seen for the noncoding strands. The binding constant for the class III promoter is (4 +/- 1.5) X 10(7) M-1 and in the presence of GTP increases to (10 +/- 1.7) X 10(7) M-1. These binding constants are about 1000 and 200 times greater, respectively, than values reported previously [Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. The differences in binding constants are probably due to tRNA and high salt used in those earlier experiments. Both tRNA and high salt (greater than 50 mM NaCl and greater than 10 mM MgCl2) inhibit the binding of the polymerase to the promoter. Optimal binding conditions occur at 2-5 mM MgCl2 and 0-10 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage T7 late promoters: construction and in vitro transcription properties of deletion mutants.

The construction of plasmids containing T7 class I promoters with deletion mutants was described. Restriction fragments, ending at the Hinf I site located at position -10 in the promoter from 14.8% of the T7 genome, were cloned into pBR322. This produced the deletion of either the left or the right part of the promoter. The in vitro transcription properties of these plasmids were determined. Control plasmids were obtained by cloning wild type class II and class III promoters into pBR322. These plasmids also were used to compare the in vitro transcription properties of the two classes of late promoters. Much of the leftward part of a T7 late promoter can be deleted without abolishing activity, but deletion of the right part eliminates promoter activity. Class II, class III, and the mutated promoters have characteristic responses to changes in ionic strength, exogenous glycerol, and temperature.

Comparison and reliability of techniques for measuring leakage and marginal penetration.

A new method for assessment of leakage and marginal penetration was developed. The new electrochemical technique employs zero resistance ammetry, and rapid quantitative results can be obtained over a long period of time. The purpose of the study was to compare the reliability of this method of assessment of leakage versus the autoradiographic and dye penetration techniques. The electrochemical data showed that there is a very sharp increase in the penetration during the first 10 days and that maximum leakage occurred on days 11 to 14 of immersion, after which electric flow remained constant. The correlation of the electric readings with the evaluations obtained with the dye penetration and autoradiographic technique was r = 0.46 and r = 0.52, respectively. The correlation was especially correct at the two extreme ends of the electric score ranges.