Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes.

Many Bacteroides clinical isolates contain large conjugative transposons, which excise from the genome of a donor and transfer themselves to a recipient by a process that requires cell-to-cell contact. It has been suggested that the transfer intermediate of the conjugative transposons is a covalently closed circle, which is transferred by the same type of rolling circle mechanism used by conjugative plasmids, but the transfer origin of a conjugative transposon has not previously been localized and characterized. We have now identified the transfer origin (oriT) region of one of the Bacteroides conjugative transposons, TcrEmr DOT, and have shown that it is located near the middle of the conjugative transposon. We have also identified a 16-kbp region of the conjugal transposon which is necessary and sufficient for conjugal transfer of the element and which is located near the oriT. This same region proved to be sufficient for mobilization of coresident plasmids and unlinked integrated elements as well as for self-transfer, indicating that all of these activities are mediated by the same transfer system. Previously, we had reported that disruption of a gene, rteC, abolished self-transfer of the element. rteC is one of a set of rte genes that appears to mediate tetracycline induction of transfer activities of the conjugative transposons. On the basis of these and other data, we had proposed that RteC activated expression of transfer genes. We have now found, however, that when the transfer region of TcrEmr DOT was cloned as a plasmid that did not contain rteC and the plasmid (pLYL72) was tested for transfer out of a Bacteroides strain that did not have a copy of rteC in the chromosome, the plasmid was self-transmissible without tetracycline induction. This and other findings suggest that RteC is not an activator transfer genes but is stimulating transfer in some other way.     

The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.     

The size distribution of insertions and deletions in human and rodent pseudogenes suggests the logarithmic gap penalty for sequence alignment

The size distributions of deletions, insertions, and indels (i.e., insertions or deletions) were studied, using 78 human processed pseudogenes and other published data sets. The following results were obtained: (1) Deletions occur more frequently than do insertions in sequence evolution; none of the pseudogenes studied shows significantly more insertions than deletions. (2) Empirically, the size distributions of deletions, insertions, and indels can be described well by a power law, i.e., f _k = Ck ^–b , where f _k is the frequency of deletion, insertion, or indel with gap length k, b is the power parameter, and C is the normalization factor. (3) The estimates of b for deletions and insertions from the same data set are approximately equal to each other, indicating that the size distributions for deletions and insertions are approximately identical. (4) The variation in the estimates of b among various data sets is small, indicating that the effect of local structure exists but only plays a secondary role in the size distribution of deletions and insertions. (5) The linear gap penalty, which is most commonly used in sequence alignment, is not supported by our analysis; rather, the power law for the size distribution of indels suggests that an appropriate gap penalty is w _k = a + b ln k, where a is the gap creation cost and blnk is the gap extension cost. (6) The higher frequency of deletion over insertion suggests that the gap creation cost of insertion (a _i ) should be larger than that of deletion (a _d ); that is, a _i – a _d = In R, where R is the frequency ratio of deletions to insertions. Correspondence to: W.-H. Li     

Pertussis Toxin-Sensitive G Protein α-Subunits: Production of Monoclonal Antibodies and Detection of Differential Increases on Differentiation of PC12 and LA-N-5 Cells

Abstract: Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein α-subunits present in mammalian brain—αo, αi1, and αi2—using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant αi2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein α-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of αi1 but not αi2 increased during nerve growth factor-induced differentiation. In contrast, αi2 but not αi1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of αo increased in both cell lines during differentiation. Electrophoretic resolution of αo subtypes revealed that although αo2 was the major subtype in undifferentiated cells, only the concentration of αo1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with αo, increased similarly to that of αo1. ADP-ribosylation of αo, αi1, and αi2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of αq, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of αi and αo associated with neuronal differentiation.     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.     

Peptides in membranes: Helicity and hydrophobicity

Synthetic model membrane-interactive peptides—both of natural and designed sequence—have become convenient and systematic tools for determination of how the membrane-spanning segments within integral membrane proteins confer protein structure and biology. Conformational studies on these peptides demonstrate that the α-helix is the natural choice of conformation for a peptide segment in a membrane, and that a helical conformation will arise “automatically” in a peptide above a threshold hydrophobicity that allows it to associate stably with the membrane. Environmental and sequential contexts thus impart conformational versatility to many of the amino acids, thereby providing a mechanism for producing the diverse structural and functional properties of proteins. © 1994 John Wiley & Sons, Inc.     

Nonlinear sequence-dependent structure of nigral dopamine neuron interspike interval firing patterns.

Firing patterns of 15 dopamine neurons in the rat substantia nigra were studied. These cells alternated between two firing modes, single-spike and bursting, which interwove to produce irregular, aperiodic interspike interval (ISI) patterns. When examined by linear autocorrelation analysis, these patterns appeared to reflect a primarily stochastic or random process. However, dynamical analysis revealed that the sequential behavior of a majority of these cells expressed "higher-dimensional" nonlinear deterministic structure. Dimensionality refers to the number of degrees of freedom or complexity of a time series. Bursting was statistically associated with some aspects of nonlinear ISI sequence dependence. Controlling for the effects of nonstationarity substantially increased overall predictability of ISI sequences. We hypothesize that the nonlinear deterministic structure of ISI firing patterns reflects the neuron's response to coordinated synaptic inputs emerging from neural circuit interactions.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.Editor's commentsThe presence of non-detectable alleles for VNTR loci has plagued the use of these highly-discriminating systems in human identification. The authors take explicit account of these alleles, and are able to show independence of the frequencies of detectable alleles. They raise the troubling issue of how to account for occasional significant results when multiple tests are performed. By invoking Bonferroni corrections, they regard all tests, even those performed on different loci, as addressing the same hypothesis—the absence of dependence at any VNTR locus.