Compartmentalization of gene expression during sporulation of Bacillus subtilis is compromised in mutants blocked at stage III of sporulation

Mutations in the spoIIIA and spoIIIJ loci disrupt the compartmentalization of gene expression during sporulation of Bacillus subtilis. The breakdown in compartmentalization is not the cause of their being blocked in spore formation. Rather, it appears to be a consequence of the engulfed prespore's being unstable.     

Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.     

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.     

Caste‐biases in gene expression are specific to developmental stage in the ant Formica exsecta

Understanding how a single genome creates and maintains distinct phenotypes is a central goal in evolutionary biology. Social insects are a striking example of co‐opted genetic backgrounds giving rise to dramatically different phenotypes, such as queen and worker castes. A conserved set of molecular pathways, previously envisioned as a set of ‘toolkit’ genes, has been hypothesized to underlie queen and worker phenotypes in independently evolved social insect lineages. Here, we investigated the toolkit from a developmental point of view, using RNA‐Seq to compare caste‐biased gene expression patterns across three life stages (pupae, emerging adult and old adult) and two female castes (queens and workers) in the ant Formica exsecta. We found that the number of genes with caste‐biased expression increases dramatically from pupal to old adult stages. This result suggests that phenotypic differences between queens and workers at the pupal stage may derive from a relatively low number of caste‐biased genes, compared to higher number of genes required to maintain caste differences at the adult stage. Gene expression patterns were more similar among castes within developmental stages than within castes despite the extensive phenotypic differences between queens and workers. Caste‐biased expression was highly variable among life stages at the level of single genes, but more consistent when gene functions (gene ontology terms) were investigated. Finally, we found that a large part of putative toolkit genes were caste‐biased at least in some life stages in F. exsecta, and the caste‐biases, but not their direction, were more often shared between F. exsecta and other ant species than between F. exsecta and bees. Our results indicate that gene expression should be examined across several developmental stages to fully reveal the genetic basis of polyphenisms.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Bcl10 is an essential regulator for A20 gene expression

A20, a tumor suppressor in several types of lymphomas, has been suggested to be an nuclear factor kappa B (NF-κB) target gene; conversely, the deubiquitylation activity of A20 is required for inhibition of Bcl10-mediated activation of NF-κB. BCL10, which is activated in a recurrent chromosomal translocation that causes human mucosa-associated lymphoid tissue lymphomas, is known to be essential for NF-κB activation in B cells. We report here that Bcl10 upregulates endogenous A20 gene expression in B lymphocytes upon B-cell receptor engagement of anti-IgM. Transient transfection assays in HEK 293 cells indicate that Bcl10 can activate the A20 promoter, which contains NF-κB-binding sites. We also construct a theoretical structure of mouse Bcl10 and analyze the structure by molecular modeling and molecular dynamics simulation. Lastly, we found that marginal zone B cells from BCL10-transgenic mice proliferate more readily than wild-type B cells, whereas, surprisingly, the transgenic follicular B cells from these mice proliferate comparably to wild-type cells. Collectively, our results indicate that Bcl10 is an essential regulator of A20 gene expression and B-cell proliferation mediated by B-cell receptor signaling.     

SV40 chromatin structure is not essential for viral gene expression

The biological activity and the fate of SV40 DNA (minichromosomes, DNA I, DNA II, DNA III) were tested in culture cells by immunofluorescence staining and blot analysis. Following microinjection of 2-4 circular SV40 molecules (minichromosomes, DNA I, DNA II) into the cytoplasm or the nuclei of monkey and rat cells, T- and V-antigen synthesis was demonstrable in nearly every recipient cell. Only linear DNA induced T-antigen synthesis with a very low efficiency after cytoplasmic injection. This low activity correlates with a rapid degradation of DNA III in the recipient cells. Further modifications observed immediately after injection are relaxation of superhelical molecules and formation of high-Mr DNA. Assembly of the injected DNA into SV40 chromatin-like structure, however, occurred only late after early viral gene expression.     

Protein kinase activity of the insulin receptor is essential for insulin-regulated gene expression

Two Chinese hamster ovary (CHO) cell lines stably transfected with human insulin receptor cDNA, CHO-wt and CHO-mut, which express an equivalent number of normal and kinase-defective human insulin receptors, respectively, were used to assess the roles of insulin receptor tyrosine kinase activity in insulin-regulated gene expression. The effect of insulin on gene-33-promoter-driven chloramphenicol acetyltransferase (CAT), RSVLTR-driven -galactosidase (pRSVLTR-gal) and SV40 late-promoter-driven hepatitis B surface antigen (pMLSV₂HBsAg) were examined in CHO-wt and CHO-mut cells. Insulin-stimulated gene 33 promoter is 10- to 50-fold more effective in CHO-wt cells than that in parental CHO cells. However, no enhancement of insulin sensitivity of gene 33 promoter in CHO-mut cells relative to parental CHO cells was found. Similar phenomena were also observed, in that insulin regulated pRSVLTR-gal and pMLSV₂HBsAg in these three CHO lines. Our data indicated that the protein kinase activity of the insulin receptor is essential for the stimulatory activity of insulin toward the activities of different promoters.     

The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response.

The Saccharomyces cerevisiae gene YPT1 encodes a protein that exhibits significant homology to the mammalian ras proteins. Using gene disruption techniques, we have shown that the intact YPT1 gene is required for spore viability. Lethality caused by loss of YPT1 function, unlike that caused by loss of the yeast ras homologs RAS1 and RAS2 function, is not suppressed by the bcy1 mutation, suggesting that YPT1 does not act through the adenylate cyclase regulatory system. A cold-sensitive allele, ypt1-1, was constructed. At the nonpermissive temperature, mutants died, exhibiting aberrant nuclear morphology, as well as abnormal distribution of actin and tubulin. The mutant cells died without exhibiting classical cell-cycle-specific arrest; nevertheless, examination of cellular DNA content suggests that the YPT1 function is required, particularly after S phase. Cells carrying the ypt1-1 mutation died upon nitrogen starvation even at a temperature permissive for growth; diploid cells homozygous for ypt1-1 did not sporulate. The YPT1 gene is thus involved in nutritional regulation of the cell cycle as well as in normal progression through the mitotic cell cycle.     

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.     

Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation.

Cell differentiation is associated either with a complete loss of proliferative potential or with a change in growth requirements. Neoplastic transformation may result from the activation of oncogenes that support growth or from inactivation or loss of tumor suppressor genes, which are thought to regulate differentiation. To examine the relationship between tumor suppressor genes and cell differentiation, we chose the gene "deleted in colorectal cancer" (DCC) and studied its role in a pheochromocytoma cell line, PC-12, using antisense RNA as well as antisense oligonucleotides to DCC. When exposed to nerve growth factor for several days, PC-12 cells develop long dendrites. This morphological change follows the transient expression of immediate early genes and is associated with an up-regulation of DCC. Interestingly, if the up-regulation of DCC was counteracted using an antisense RNA technique, the morphological changes were prevented, but the other parameters of the nerve growth factor response were unaffected. Moreover, when DCC expression was inhibited by antisense oligonucleotides to DCC in nerve growth factor-differentiated cells, the neuron-like phenotype was reversed. Our results demonstrate that the gene DCC is involved in a distal segment of neural differentiation and provide the first direct evidence that a tumor suppressor gene plays a role in cell differentiation.     

cDNA cloning and developmental expression of the porcine homologue of WT1

Wilms' tumors occur most frequently in swines as sporadic tumors. To clarify the role of WT1 gene in the genesis of Wilms' tumors and genitourinary development, we have isolated the porcine homologue of the human WT1 gene (pWT1) and analyzed its expression in various organs including the kidney. The open reading frame of pWT1 cDNA was extremely homologous to the human counterpart: 94% identical at the nucleotide level and 98% at the polypeptide level. In particular, the zinc finger region was more than 97% similar to human WT1 gene at the nucleotide level and 100% at the polypeptide level. pWT1 mRNA was found to be expressed in new-born kidney, spleen, testis, and embryonic kidneys, suggesting a possible association of pWT1 with the development of the genitourinary system. In conclusion, the nucleotide sequence and expression patterns in organs of pWT1 were similar to those of human WT1. Therefore, swines could provide good models for analyzing the contributions of WT1 gene to genitourinary development and genesis of Wilms' tumors.