Nutrients and heavy metal contamination of plants and sediments in Futian mangrove forest

An ecological survey was carried out to determine the levels of nutrients and heavy metals in the sediments and leaf tissues of two dominant mangrove plant species, Kandelia candel and Aegiceras corniculatum, in Futian mangrove forest, Shenzhen, the People's Republic of China. The spatial and seasonal variations of these elements were also investigated. The results show that there was no major difference between two sampling sites 150 m apart. In both sites, the sediment concentrations of total and NH₄ ⁺-N, total and extractable P, total and extractable K, total organic carbon were consistently higher in the landward locations and decreased gradually towards the sea. The sediment sample collected at the seaward edge of the mangrove plant community had the lowest levels of nutrient and organic matter. The vertical variations (from the land to the sea) of sediment heavy metals were less obvious and no particular trend could be identified. Extremely high contents of Cu, Cd, Pb, Cr and Zn were found at certain locations, suggesting the occurrence of some local contamination. The mean total metal concentrations in sediments decreased in the order Mn > Zn > Cu > Cr = Pb > Cd for the sample locations. Most of the heavy metals were not in a bioavailable form as the concentrations of extractable metals were relatively low (< 1% of total metals). Pb, Cr and Cd were not detected in leaf samples. Leaf C, N, P and K contents were similar between the two species and no significant difference was found among locations, although A. corniculatum seemed to have lower Mn concentrations than K. candel. With reference to temporal variations, no significant difference in sediment concentrations of some nutrients and metals was found between the spring and autumn seasons.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

Mechanics of bacterial macrofiber initiation.

The twisting and writhing during growth of single-cell filaments of Bacillus subtilis which lead to macrofiber formation was studied in both left- and right-handed forms of strains FJ7 and RHX. Filament bending, touching, and loop formation (folding), followed by winding up into a double-strand fiber, were documented. Subsequent folds that produced multistrandedness were also examined. The rate of loop rotation during winding up was measured for 26 loops from 16 clones. In most cases, the first loop formed turned at a lower rate than those produced by the following cycles of folding. The sequence of folding topologies differed in FJ7 and RHX strains and in left- versus right-handed structures. Right-handed FJ7 routinely gave rise to four-stranded helices at the second fold, whereas left-handed FJ7 and both left-handed and right-handed forms of RHX made structures with predominantly two double-stranded helical regions. Left-handed RHX structures frequently produced second folds within the initial loop itself, resulting in T- or Y-shaped fibers. Sixteen cases in which the initial touch of a filament to itself produced a loop that snapped open before it could wind up into a double-strand fiber were found. The snap motions were used to obtain estimates of the forces generated by helical growth of single filaments and to investigate theoretical models involving the material properties of cell filaments. In general, the mechanical behavior of growing single-cell filaments and fibers consisting of two-, three-, or four-strand helices was similar to that described for larger, mature, multifilament macrofibers. The behavior of multicellular macrofibers can be understood, therefore, in terms of individual cell growth.     

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.     

Estimating the intensity of male-driven evolution in rodents by using X-linked and Y-linked Ube 1 genes and pseudogenes

Using sequence data from the last introns of ZFX and ZFY genes, we previously estimated the male-to-female ratio () of mutation rate to be close to 6 in higher primates and 1.8 in rodents. As the mutation rate may vary among different regions of the mammalian genome, it is interesting to see whether sequence data from other regions will give similar estimates. In this study, we have determined the partial genomic sequences of the ubiquitin-activating enzyme El genes (Ube 1x and Ube 1y for the X-linked and Y-linked homologues, respectively) of mice and rats and two mouse Ube 1y pseudogenes. From the intron sequences of the Ube 1 genes, we calculated the divergence of the Y-linked genes (Y = 0.161) and that of the X-linked genes (X = 0.107) between mouse and rat, and found the Y/X ratio to be 1.50. This ratio led to an estimate of = 2.0 with a 95% confidence interval of (1.0, 3.9). Similar estimates of were obtained if mouse Ube 1y pseudogenes were used instead of the mouse Ube 1y functional gene. These estimates are consistent with our previous estimate for rodents and suggest that the sex ratio of mutation rate in rodents is approximately only one-third of that in higher primates. Our estimate of the divergence time between Ube 1x and Ube 1y supports the view that the two genes separated before the eutherian radiation.Correspondence to: W.-H. Li     

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.     

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.     

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.