Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification

The alternate expression of the Salmonella flagellin genes H1 and H2 is controlled by the orientation of a 995-base-pair invertible segment of DNA located at the 5' end of the H2 gene. The hin gene, which is encoded within the invertible region, is essential for the inversion of this DNA segment. We cloned the hin gene into Escherichia coli and placed it under the control of the PL promoter of bacteriophage lambda. These cells overproduced the Hin protein. In vivo inversion activity was measured by using a recombinant lambda phage which contains the H2 and lacZ genes under the control of the invertible region. Using this phage, we showed that the amount of inversion activity is proportional to the amount of Hin protein in the cell. An inactive form of the protein was purified by using the unusual solubility properties of the overproduced protein. The amino acid composition of the protein agreed with the DNA sequence of the hin gene. Antibodies were made to the isolated protein. These antibodies cross-reacted with two other unidentified E. coli proteins

Why a Particle Physicist is Interested in DNA Branch Migration

We describe an explicitly discrete model of the process of DNA branch migration. The model matches the existing data well, but we find that branch migration along long strands of DNA (N \simge 40~bp) is also well modeled by continuum diffusion. The discrete model is still useful for guiding future experiments.Comment: Talk presented at LATTICE96(theoretical developments); 3 pages, TeXsis w/ LAT96.txs (available from ftp://lifshitz.ph.utexas.edu/texsis/styles/LAT96.txs and will be a part of the next Elsevier.txs) and TXSdcol.te

Secondary structural characterization of oligonucleotide strands using electrospray ionization mass spectrometry

Differences in charge state distributions of hairpin versus linear strands of oligonucleotides are analyzed using electrospray ionization mass spectrometry (ESI-MS) in the negative ion detection mode. It is observed that the linear structures show lower charge state distribution than the hairpin strands of the same composition. The concentration of ammonium acetate and the cone voltage are major factors that cause the shift of the negative ions in the charge states. The ESI data presented here are supported by UV spectra of strands acquired at 260 nm wavelength in aqueous ammonium acetate solution. We will show that the strands that demonstrate a higher charge state distribution in the gas phase also have a higher melting temperature in solution

The relationship between parenting, family interaction and childhood dental caries: A case-control study

a b s t r a c t The aim of this case-control study was to explore the relationship between parenting practices, parent echild interaction and childhood dental caries, using a sample of 5e8-year old children from the Netherlands. Cases were defined as children with four or more decayed, missing or filled teeth and controls were caries free. Cases (n ¼ 28) and controls (n ¼ 26) were recruited from a referral centre for paediatric dental care and a general dental practice, respectively. Parenting practices and parentechild interactions of the child's primary caregiver were observed using Structured Interaction Tasks and subsequently rated on seven dimensions: positive involvement, encouragement, problem-solving, discipline, monitoring, coercion and interpersonal atmosphere. All Structured Interaction Tasks were videotaped, and coded by trained and calibrated observers blind to the dental condition. Differences in parenting dimensions between cases and controls were analysed using multivariate analysis of variance, independent samples T-tests, c 2 -tests and multiple logistic regression analyses. Controls had significantly higher scores on the dimensions positive involvement, encouragement, problem-solving and interpersonal atmosphere, compared to cases. Parents of controls were also less likely to show coercive behaviours. These associations remained statistically significant after adjustment for the mother's education level, tooth brushing frequency and the frequency of consuming sugary foods and drinks, except for coercion. There was no significant difference in discipline between cases and controls. In conclusion, this case-control study found a significant relationship between parenting practices, parent echild interaction quality and childhood dental caries. Our findings suggest that parenting practices may be an important factor to consider in caries preventive programs

The M\u3csub\u3er\u3c/sub\u3e-value of chloroplast coupling factor 1

The Mr of spinach chloroplast coupling factor 1 has been determined by sedimentation equilibrium and by light scattering to be 400 000 ± 24 600 and 407 000 ± 20 000, respectively. These values differ substantially from that obtained previously (325 000) and are consistent with an α3β3γδε{lunate} subunit stoichiometry. © 1983

Regulatory Cross-Talk Links Vibrio cholerae Chromosome II Replication and Segregation

There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains—one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation

DNA-binding properties of the Hin recombinase

The recombinase of the Salmonella inversion system, Hin, mediates site-specific recombination between two 26 base pairs (bp) inverted repeat sequences (hixL and hixR) which flank a 993-bp DNA segment. We have investigated Hin recognition of, and association with, the hix recombination sites. Nuclease and chemical protection studies with linear and supercoiled DNA substrates demonstrate that Hin initially binds hixL and hixR independently of binding of the other protein components of the inversion system, Fis and HU. DNA-binding assays with mutant recombination sites and methylation interference experiments indicate that the critical bases for Hin recognition of its DNA-binding site are within an 8-bp sequence covering adjacent major and minor grooves of the DNA helix in each of the 12-bp half-sites of the hix recombination sites. The nature of the Hin-hix complexes in these binding studies and the results of gel filtration assays with purified Hin suggests that Hin binds the recombination sites as a dimer. The implications of the nature of the interactions of Hin with its recombination sites on the mechanism of the recombination reaction and on the novel features of DNA recognition by Hin are discussed