Multiple N-cadherin enhancers identified by systematic functional screening indicate its Group B1 SOX-dependent regulation in neural and placodal development

Neural plate and sensory placodes share the expression of N-cadherin and Group B1 Sox genes, represented by Sox2. A 219-kb region of the chicken genome centered by the N-cadherin gene was scanned for neural and placodal enhancers. Random subfragments of 4.5 kb average length were prepared and inserted into tkEGFP reporter vector to construct a library with threefold coverage of the region. Each clone was then transfected into N-cadherin-positive (lens, retina and forebrain) or -negative embryonic cells, or electroporated into early chicken embryos to examine enhancer activity. Enhancers 1-4 active in the CNS/placode derivatives and non-specific Enhancer 5 were identified by transfection, while electroporation of early embryos confirmed enhancers 2-4 as having activity in the early CNS and/or sensory placodes but with unique spatiotemporal specificities. Enhancers 2-4 are dependent on SOX-binding sites, and misexpression of Group B1 Sox genes in the head ectoderm caused ectopic development of placodes expressing N-cadherin, indicating the involvement of Group B1 Sox functions in N-cadherin regulation. Enhancers 1, 2 and 4 correspond to sequence blocks conserved between the chicken and mammalian genomes, but enhancers 3 and 5 are unique to the chicken.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168

Summary A localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Response Characteristics between Sulfate-Reducing Bacteria and Environmental Factors in Petroleum Hydrocarbon-Contaminated Field of Northeast China

Lithology samples were collected from six sites of a petroleum hydrocarbon-contaminated site in northeast China along a contamination plume. The sulfate-reducing bacteria (SRB) diversity of all samples was analyzed by PCR-DGGE technology. The Shannon-Wiener indexes (1.004–3.665), Simpson indexes (0.516–0.907) and Pielou indexes (0.996–1.004) of all samples were used to characterize the abundances, advantages and evenness of the microbial communities. Additionally, Canoco for Windows 4.5 was utilized to analyze the correlation between dominant SRB and environmental factors. The results showed that the abundance, advantages and evenness of the sulfate-reducing bacterial community changed regularly along the contamination plume direction. The microbial homology of the samples was not high (0.25–0.80), and the dominant bacteria exhibited heteroplasmy. Additionally, the dominant bacteria were identified as uncultured bacteria. Canonical correspondence analysis (CCA) analysis showed that the distribution and structure of SRB communities were not obviously correlated with the concentration of total petroleum hydrocarbons (TPH), dissolved oxygen (DO) and other environmental factors. The results presented herein provide evidence of natural bioremediation in petroleum hydrocarbon-contaminated fields and information that will be useful to bioremediation of other contaminated fields.     

Characterization of the N-terminal structure of pertussis toxin subunit S1 and hybridization of oligodeoxyribonucleotide probes with a Bordetella pertussis DNA fragment

Abstract N-terminal amino acid sequence analysis of the enzymatic subunit S1 of pertussis toxin from Bordetella pertussis showed the structure of the first 25 residues. 2 different oligodeoxyribonucleotide probes were synthesized as mixed 32-mers corresponding to the DNA sequence deduced. Southern blot analysis of pertussis DNA digested with restriction endonuclease Cla I showed that both mixed probes hybridized with a DNA fragment of about 10 kb, thus identifying a B. pertussis gene corresponding to the protein structure determined.