Characterization of two Drosophila melanogaster cytochrome c genes and their transcripts.

Analysis of total Drosophila melanogaster DNA by genomic blot hybridization indicates that two cytochrome c-like sequences exist in the Drosophila genome. These two sequences, DC3 and DC4, have been isolated from a Charon 4A-D. melanogaster genomic library. DC3 and DC4 are located within a 4 kb region of DNA, at position 36A 10-11, on the left arm of chromosome 2. The nucleotide sequence of these two clones has been determined. Both DC3 and DC4 can encode functional cytochrome c proteins. The polypeptide sequences predicted by these two genes, however, differ at 32 amino acid residues. DC4 is expressed at varying, but relatively high levels throughout Drosophila development. In contrast, DC3 is expressed at constant, but relatively low levels throughout development.     

Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B

Hep 3B, a human hepatoma cell line was examined for its RNA hybridizable to the hepatitis B virus sequence. Using probes that covered different regions of the hepatitis B virus genome, five species of RNA were observed of sizes 4.0, 3.3, 2.9, 2.6 and 2.2 kilobases. The RNAs covered surface antigen gene, pre-S and X regions. None of them had a core antigen sequence. RNA with a 4.0 kilobase size was the most abundant. Using S1 nuclease analysis, its 5' end of hepatitis B virus sequence was mapped at pre-S region and its 3' end of viral sequence was mapped at DR region.     

Arabidopsis CUL3A and CUL3B genes are essential for normal embryogenesis

Cullin (CUL)-dependent ubiquitin ligases form a class of structurally related multisubunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. The CUL3-BTB ligases belong to this class of enzymes and despite recent findings on their molecular composition, our knowledge on their functions and substrates remains still very limited. In contrast to budding and fission yeast, CUL3 is an essential gene in metazoans. The model plant Arabidopsis thaliana encodes two related CUL3 genes, called CUL3A and CUL3B. We recently reported that cul3a loss-of-function mutants are viable but exhibit a mild flowering and light sensitivity phenotype. We investigated the spatial and temporal expression of the two CUL3 genes in reproductive tissues and found that their expression patterns are largely overlapping suggesting possible functional redundancy. Thus, we investigated the consequences on plant development of combined Arabidopsis cul3a cul3b loss-of-function mutations. Homozygous cul3b mutant plants developed normally and were fully fertile. However, the disruption of both the CUL3A and CUL3B genes reduced gametophytic transmission and caused embryo lethality. The observed embryo abortion was found to be under maternal control. Arrest of embryogenesis occurred at multiple stages of embryo development, but predominantly at the heart stage. At the cytological level, CUL3 loss-of-function mutations affected both embryo pattern formation and endosperm development.     

Genetic analysis of Escherichia coli urease genes: evidence for two distinct loci.

Studies with two uropathogenic urease-producing Escherichia coli strains, 1021 and 1440, indicated that the urease genes of each are distinct. Recombinant plasmids encoding urease activity from E. coli 1021 and 1440 differed in their restriction endonuclease cleavage sites and showed minimal DNA hybridization under stringent conditions. The polypeptides encoded by the DNA fragments containing the 1021 and 1440 urease loci differed in electrophoretic mobility under reducing conditions. Regulation of urease gene expression differed in the two ureolytic E. coli. The E. coli 1021 locus is probably chromosomally encoded and has DNA homology to Klebsiella, Citrobacter, Enterobacter, and Serratia species and to about one-half of the urease-producing E. coli tested. The E. coli 1440 locus is plasmid encoded; plasmids with DNA homology to the 1440 locus probe were found in urease-producing Salmonella spp., Providencia stuartii, and two E. coli isolates. In addition, the 1440 urease probe was homologous to Proteus mirabilis DNA.     

Degradation of MUC7 and MUC5B in Human Saliva

Background

Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis.

Methodology/Principal Findings

Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours.

Conclusions/Significance

MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.     

A method for simultaneous detection of the transcripts of different cellular genes

In this paper, a method for preparing radioactive-labelled first-strand cDNA has been described and the major procedures and reaction conditions have been studied. Some advantages provided by this method for simultaneously detecting the expression of multiple cellular genes are discussed.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

Cairnsian mutagenesis inEscherichia coli: Genetic evidence for two pathways regulated bymutS andmutL genes

The phenomenon of Cairnsian mutagenesis was studied inEscherichia coli mutants bearing mutations inmutS,mutL,recA andlexA genes. It is shown that development of resistance to exogenous valine could be used as an example of Cairnsian response. Strains defective inmutS andmutL show a high frequency of Cairnsian mutagenesis to valine resistance. The response inmutS mutants is dependent upon cleavability of the LexA protein whereas that inmutL is not. The latter is independent ofrecA also. The need for LexA protein cleavage inmutS mutants can be bypassed by over-production of the RecA protein due to arecA operator constitutive mutation. Genetic evidence is presented to show that the products ofmutS andmutL genes negativelycontrol two pathways of Cairnsian mutagenesis. Cairnsian response is also elicited whenmutS ormutL strains are grown under conditions wherein a required nutrient is present in sub-optimal concentrations. Random, unselected mutagenic events are likely to occur during or after Cairnsian mutagenesis provided the cells are SOS inducible.     

C-Mannosylation of MUC5AC and MUC5B Cys subdomains

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.