Immunization of mice against Salmonella typhimurium using different DNA preparations.

Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S. typhimurium, P. aeruginosa, or S. aureus. Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline. Mice in all groups were challenged intraperitoneally with 100 LD50 of S. typhimurium. The bacterial genomic DNA was extracted using the Pure Gene extraction kit. Specific primers were used to amplify the 1.57kb fragment by PCR. The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/ 1.57kb construct. Bacterial genomic DNA extracted from P. aeruginosa and S. aureus appeared to induce non-specific resistance in mice. Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S. typhimurium was used. There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge. None of the mice in the saline control group survived by day 7 post challenge. It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA. Specific resistance obtained when genomic DNA from S. typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.

Structure and expression of hybrid dysgenesis-induced alleles of the ovarian tumor (otu) gene in Drosophila melanogaster.

Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function.

Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression.

We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster. Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.

A specific ovarian tumor protein isoform is required for efficient differentiation of germ cells in Drosophila oogenesis.

Mutations within the ovarian tumor (otu) gene result in abnormal ovarian development. It has been proposed that otu phenotypes result from abnormal germ cell division and differentiation. To understand better what role otu performs in oogenesis we have undertaken an analysis of protein expression from the otu locus. Anti-otu antibodies recognize two proteins from Drosophila ovary extracts with apparent molecular masses of 98 and 104 kD. Sequence analysis of otu cDNAs suggests that these proteins are translated from two mRNAs generated by alternative splicing of a 126-bp exon between the sixth and seventh exon of the smaller transcript. Analysis of otu protein expression in eight mutants indicates a correlation between the accumulation of the 104-kD isoform and predifferentiated germ cells and suggests that there is a developmental shift in the accumulation of the two isoforms upon differentiation of germ cells. Furthermore, the 104-kD isoform appears to be required for efficient differentiation of germ cells. Immunostaining of otu proteins is restricted to the cytoplasm of germ cells, and a rapid loss of oocyte immunostaining during stage 11 suggests that there is a rapid and selective degradation of otu proteins within the oocyte but not within its 15 interconnected nurse cells.

Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein.

Sequencing cDNA and genomic DNA from the ovarian tumor gene revealed a gene with seven introns spanning 4.5 kilobases. The proline-rich, hydrophilic otu protein is novel. An antibody prepared to a beta-gal-otu fusion protein recognized a 110-kilodalton ovarian protein which was altered in the ovaries of otu gene mutants.

Molecular localization and developmental expression of the otu locus of Drosophila melanogaster.

The female-sterile ovarian tumor gene, otu, is located in cytological region 7F1 on the Drosophila melanogaster chromosome map. We have mapped the gene at the molecular level by using four dysgenic alleles and two revertant derivatives of these alleles as well as an ethyl methanesulfonate-induced allele. The insertional (dysgenic) changes were all associated with one restriction fragment, and its size was restored after phenotypic reversion. One ethyl methanesulfonate-induced allele had a deletion in the restriction fragment adjacent (distal) to the fragment altered in the insertional alleles. These two restriction fragments were immediately adjacent to the s38 chorion gene. Associated with the two altered restriction fragments were two RNA species, an abundant 3.2-kilobase (kb) poly(A)+ RNA and a minor 4.0-kb RNA. Several other less-abundant RNA species were detectable with more-sensitive single-stranded RNA probes. The otu gene was transcribed proximal to distal relative to the centromere; this was opposite to the direction of transcription of the adjacent s38 gene. During development, the 3.2-kb RNA was absent in larvae, first appeared in the pupal stages, and persisted in adult females, in which it was most prevalent in the ovaries. The DNA that hybridized to the 3.2-kb ovarian RNA hybridized to four different RNAs found in the testes but not in the rest of the adult male. These testis-enriched RNAs were transcribed from the same strand of DNA as the ovarian transcripts.

Amplification of the X-linked Drosophila chorion gene cluster requires a region upstream from the s38 chorion gene.

Genomic sequences controlling follicle cell-specific amplification of the X-linked Drosophila chorion gene cluster were mapped by P element-mediated transformation. Several DNA fragments containing the s38 gene and flanking sequences induced tissue-specific amplification, although replication levels were subject to position effects. Deletion analysis identified a 467-bp region upstream from the s38 transcription start site that contained sequences essential in cis for amplification. The essential region shared 32 bp of imperfect sequence homology with a previously identified region necessary for third chromosome chorion gene cluster amplification. This homologous segment contained a repetitive motif consisting of perfect and imperfect AATAC repeats; it was localized near the boundary of the essential domain since most, but not all, the repeats could be deleted without eliminating transposon-induced amplification. The repetitive region was not required for developmentally regulated s38 transcription, therefore our results identified at least one element required for amplification but not for chorion gene transcription. The homologous repetitive sequences within the amplification-essential regions may constitute part of the replication origins used to differentially replicate the two chorion domains during oogenesis.

Developmentally regulated expression of Drosophila chorion genes introduced at diverse chromosomal positions.

Drosophila chorion genes are organized into two clusters that are selectively amplified in the ovarian follicle cells. During oogenesis the transcription of individual genes is temporally regulated, resulting in distinct, stage-specific profiles of chorion mRNA accumulation. P element-mediated gene transfer was used to study the regulation of genes encoding the major chorion proteins s15-1 and s38-1. Transformed chorion genes integrated at diverse chromosomal locations exhibited proper tissue-specific and stage-specific expression, despite separation from the gene clusters. Qualitatively normal expression was not dependent on the ability of the inserted DNA to undergo amplification. However, chromosome position quantitatively influenced the RNA produced by the transformed genes. The level of RNA per gene copy produced by individual transformed genes varied approximately tenfold, after correction for differences in gene dosage due to the amplification of some inserted sequences. Transformation experiments with an s38-1-lacZ fusion gene demonstrated that cis-regulatory sequences sufficient for the stage-specific program of s38-1 expression were confined to a 1.3 X 10(3) base-pair segment between -748 and +573 relative to the s38-1 initiation site. Finally, egg chamber-specific amplification was induced at the site of two s38-1 insertions, suggesting that an amplification control element resides near this gene.

Analysis of transcriptional regulation of the s38 chorion gene of Drosophila by P element-mediated transformation.

Transcriptional regulation of the s38 chorion gene was studied using P element-mediated germline transformation. A 5.27 kb DNA fragment containing the s38 gene and 5'- and 3'-flanking sequences, was tested for its ability to be transcribed with correct developmental specificity. Five single-insert transformed lines were generated by microinjection of this DNA fragment cloned into a marked P element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native s38 gene. The 1.3 kb at the 5' end of this tested fragment was fused to the E. coli lac z gene. This fragment was also capable of initiating transcription of E. coli lac z RNA with the developmental profile of the native s38 gene. In vitro deletion studies are underway to determine which sequences in the 1.3 kb fragment are necessary for regulating the developmental expression of the gene.

Developmental regulation of Drosophila alpha-tubulin genes.

Transcripts from the four different Drosophila melanogaster alpha-tubulin genes were detected by filter hybridization experiments that used subcloned fragments from each gene as hybridization probes. These hybridization experiments demonstrated that each gene is transcribed. All of the transcripts are found on polysomes and are long enough to encode an alpha-tubulin protein. The hybridization studies were extended to examine the developmental pattern of RNA concentrations. The concentration of RNAs from the alpha 2 and alpha 4 genes vary independently and dramatically, while those from alpha 1 and alpha 3 have parallel variations. We conclude that at the RNA level of expression, two alpha-tubulin genes are regulated in parallel and two genes are not. We hypothesize that the different concentration patterns reflect different functions for the protein products of each gene.

Drosophilia alpha-tubulin genes and their transcription patterns.

There are four different alpha-tubulin genes in D. melanogaster DNA; three of them appear as single copies and the other is present as either one or two copies in the haploid genome. The transcripts of three of these genes were examined. Each of them is complementary to a transcript of different length, implying that each is transcribed. Since these transcripts are found on polysomes, it is likely that they are translated. At least two of the genes are complementary to several transcripts, indicating that each of them has more than one transcription start or stop site or perhaps that there are alternative paths of posttranscriptional processing. There is a different developmental pattern of concentrations for transcripts from each of these genes, and different RNAs from the same gene also have different patterns. We conclude that the concentration of transcripts from each gene appears to be independently controlled and that even different transcription products from the same gene appear to independently controlled.

alpha-Tubulin genes of Drosophila.

Four Drosophila alpha-tubulin genes have been isolated on recombinant DNA molecules. The identity of two of these genes (T alpha 1 and T alpha 2) was established by isolating complementary mRNAs and then examining the in vitro translation products of the mRNAs. The one- and two-dimensional gel patterns and the peptide maps of the in vitro products were indistinguishable from those of embryonic alpha-tubulin. In turn, the embryonic tubulin was identified by determining its amino-terminal sequence. We identified two other cloned alpha-tubulin genes (T alpha 3 and T alpha 4) by their complementarity to T alpha 1 and T alpha 2. Maps of restriction endonuclease sites indicate that the four genes are different. DNA hybridization studies demonstrated, however, that three of them have extensive sequence homology with each other and slight homology with the fourth, T alpha 4. Hybridization to genomic DNA fragments indicated that the four clones genes account for all of the different alpha-tubulin genes of Drosophila melanogaster. Three of them are present only once in the haploid genome; the other, T alpha 1, is present in either one or two copies. Each of the four genes hybridizes in situ to a different site on the third chromosome.

Effect of transferred adult Schistosoma mansoni on resistance of mice to cercarial challenge.

Four or eight adult Schistosoma mansoni surgically transfered to the mesenteric veins of mice survive well and may play a role in resistance to later cercarial challenge. After receiving transfers of single sex or paired flukes, animals exposed to 50or 100 cercariae showed decreased numbers of parasites derived from the challenge infection when compared with control animals 30 days later. Although statistical analysis of the data indicated that the presence of transferred S. mansoni probably had an adverse effect on the survival of worms in a subsequent challenge, the results were interpreted as inconclusive.

The host antigen phenomenon in experimental murine schistosomiasis. III. Destruction of parasites transferred from mice to hamsters.

The surgical transfer of 23- to 28-day-old Schistosoma mansoni from donor mice to the mesenteric vessels of outbred hamsters is followed by recovery of 70% of the transferred trematodes 30 days later. Prior immunization of hamsters with mouse erythrocytes results in anti-mouse erythrocyte antibody titers of greater than 1:4,096 and death of 98% of the parasites. Destruction is correlated with evidence of the presence of mouse antigenic determinants on the surfaces of schistosomes from donor mice.