Role of cAMP-Dependent Protein Kinase during Growth and Early Development of Dictyostelium discoideum

AbstractcAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIγ promoter, while expression of the discoidinIγ promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes

Identification of single nucleotide polymorphism of growth hormone gene exon 4 and intron 4 in Pesisir cattle, local cattle breeds in West Sumatera Province of Indonesia

The pupose of this study was to identify genetic polymorphisms of bovine growth hormone gene exon 4, and intron 4 in local cattle breeds in West Sumatera Province of Indonesia. DNA was isolated from 60 blood samples and polymerase chain reaction (PCR) product of GH5 fragment (366 bp) were directly sequenced. Multiple alignments, including 60 bGH DNA sequences obtained by direct sequencing and bGH sequences from a public database (National Center for Biotechnology Information, acces number M57764), revealed 15 polymorphisms (five SNP, eight deletion, and two insertion). Eight deletions were detected in position 1740, 1743, 1745, 1747, 1749, 1750, 1753, and 1754 with frequency allele of 0.50, 0.22, 0.125, 0.53, 0.06, 0.06 and 0.83, respectively. Besides, two insertions C were detected in position 1790 and 1895 with frequency allele of 0.06 and 1.00, respectively. Five mutations were detected in position 1914, 1930, 1947, 1980, and 2025 with genotypes C → G, G → A, T → G, T → C, and A → G respectively with frequency allele of 0.40, 0.48, 1.00, 0.42, and 0.38, respectively. Another deletion at position 1740, 1743, and 1754 changed codon CAG, TCG, TGG, CTT, GGG, CCC to codon CGT, GTG, GCT, TGG, GCC and new deletion at position 1745, 1749, and 1754 changed the codon TCG, TGG, CTT, GGG, CCC, CTG to TCG, GGC, TGG, GCC, CTG. Deletion at position 1749 and 1754 changed the codon TCG, TGG, CTT, GGG, CCC, and CTG to TCG, TGG, CTG, GGC, CCT. These data provide evidence that GH gene of this breed is slightly different from other breeds. This polymorphic source can be used to refer to performance and to investigate whether these polymorphics are responsible for quantitative variation in growth.Keywords: Sapi Pesisir, direct sequencing, growth hormone gene, polymorphismAfrican Journal of Biotechnology Vol. 12(3), pp. 249-25

HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing

We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the ‘dicer-related-helicase’ from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF(−) cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF(−) cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways

Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is ∼0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing