RNA-mediated gene silencing.

A number of gene-silencing phenomena including co-suppression discovered in plants, quelling in fungi and RNA interference in animals have been revealed to have steps in common. All occur in the cytoplasm at a post-transcriptional level with the mRNAs of target genes degraded in a sequence-specific manner. Small non-coding RNA molecules demonstrated to be mediators of these silencing phenomena have also been shown to mediate a parallel post-transcriptional gene silencing (PTGS) mechanism that regulates the expression of developmental genes, although in this latter mechanism, rather than being degraded, the translation of target mRNAs is inhibited. Both types of small RNA appear to be processed from longer double-stranded RNAs (dsRNAs) by a common endonuclease. RNAs may also operate as regulators of gene expression at a transcriptional level in the nucleus, via chromatin remodelling or RNA-directed DNA methylation. Methylation of promoter sequences leads to transcriptional gene silencing, while methylation of coding sequences by the same homology-dependent mechanism does not block transcription, but leads to PTGS. In some organisms, the RNA silencing signal may spread to other tissues inducing systemic RNA silencing.

The QDE-3 homologue RecQ-2 co-operates with QDE-3 in DNA repair in Neurospora crassa.

The post-transcriptional gene silencing mechanism in Neurospora crassa, called quelling, was shown to involve the products of three genes termed quelling-defective. A homologue to the qde-3 gene encoding a putative RecQ-type DNA helicase was isolated and was named RecQ-2. Characterisation of the RecQ-2 gene has revealed that it is not involved in quelling, but may co-operate with the qde-3 gene product in a pathway that repairs damage to DNA caused by the chemical mutagens methyl methanesulfonate and N-methyl- N'-nitro- N-nitrosoguanidine. These results indicate that the qde-3 RecQ helicase may have a dual role in N. crassa, either acting alone as an essential component of the quelling mechanism or together with the RecQ-2 RecQ helicase, as part of a process to repair DNA lesions during replication.

Homology-dependent gene silencing mechanisms in fungi.

Homology-dependent gene silencing (HDGS) is a ubiquitous phenomenon among fungi, plants, and animals. Gene silencing can be triggered and can affect artificially introduced nucleic acid molecules, both DNA and RNA, and/or can act on endogenous duplicated sequences. Although the various HDGS phenomena may be related each other, probably deriving from an ancestral defense mechanism, relevant differences do exist between different HDGS mechanisms. Especially in fungi, a variety of HDGS phenomena have been uncovered during the past 10 years: Gene inactivation of duplicated sequences can be achieved either through DNA-methylation and block of transcription or through sequence-specific degradation of mRNA. Moreover, duplicated sequences can also be specifically mutagenized. Studying HDGS in fungi gives us the opportunity to study such complex mechanisms in relatively simple organisms in which both genetic and biochemical approaches can be easily used.

Post-transcriptional gene silencing across kingdoms.

Post-transcriptional gene silencing (PTGS) as a consequence of the introduction of either transgenes or double-stranded RNA molecules has been found to occur in a number of species. In the past year, studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena. The ubiquitous presence of PTGS in both the plant and animal kingdoms and the finding of common genetic mechanisms suggest that PTGS is a universal gene-regulation system fundamental in biological processes such as protection against viruses and transposons.

Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase.

The phenomenon of posttranscriptional gene silencing (PTGS), which occurs when a transgene is introduced into a cell, is poorly understood. Here, the qde-3 gene, which is required for the activation and maintenance of gene silencing in the fungus Neurospora crassa, was isolated. Sequence analysis revealed that the qde-3 gene belongs to the RecQ DNA helicase family. The QDE3 protein may function in the DNA-DNA interaction between introduced transgenes or with an endogenous gene required for gene-silencing activation. In animals, genes that are homologous to RecQ protein, such as the human genes for Bloom's syndrome and Werner's syndrome, may also function in PTGS.

Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme.

Homology-dependent gene silencing is a phenomenon that occurs in a broad range of organisms and has implications for both basic and applied science. Gene silencing is a mechanism that controls invading transposons and provides protection against virus infections. It also has evolutionary implications in genome maintenance. Recent studies have begun to unravel the molecular mechanisms of this puzzling phenomenon.

Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.

In plants and fungi, the introduction of transgenes can lead to post-transcriptional gene silencing. This phenomenon, in which expression of the transgene and of endogenous genes containing sequences homologous to the transgene can be blocked, is involved in virus resistance and genome maintenance. Transgene-induced gene silencing has been termed quelling in Neurospora crassa and co-suppression in plants. Quelling-defective (qde) mutants of N. crassa, in which transgene-induced gene silencing is impaired, have been isolated. Here we report the cloning of qde-1, the first cellular component of the gene-silencing mechanism to be isolated, which defines a new gene family conserved among different species including plants, animals and fungi. The qde-1 gene product is similar to an RNA-dependent RNA polymerase found in the tomato. The identification of qde-1 strongly supports models that implicate an RNA-dependent RNA polymerase in the post-transcriptional gene-silencing mechanism. The presence of qde-1 homologues in a variety of species of plants and fungi indicates that a conserved gene-silencing mechanism may exist, which could have evolved to preserve genome integrity and to protect the genome against naturally occurring transposons and viruses.

Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa.

We report the isolation of 15 Neurospora crassa mutants defective in "quelling" or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.

Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation.

The molecular mechanisms involved in transgene-induced gene silencing ('quelling') in Neurospora crassa were investigated using the carotenoid biosynthetic gene albino-1 (al-1) as a visual marker. Deletion derivatives of the al-1 gene showed that a transgene must contain at least approximately 132 bp of sequences homologous to the transcribed region of the native gene in order to induce quelling. Transgenes containing only al-1 promoter sequences do not cause quelling. Specific sequences are not required for gene silencing, as different regions of the al-1 gene produced quelling. A mutant defective in cytosine methylation (dim-2) exhibited normal frequencies and degrees of silencing, indicating that cytosine methylation is not responsible for quelling, despite the fact that methylation of transgene sequences frequently is correlated with silencing. Silencing was shown to be a dominant trait, operative in heterokaryotic strains containing a mixture of transgenic and non-transgenic nuclei. This result indicates that a diffusable, trans-acting molecule is involved in quelling. A transgene-derived, sense RNA was detected in quelled strains and was found to be absent in their revertants. These data are consistent with a model in which an RNA-DNA or RNA-RNA interaction is involved in transgene-induced gene silencing in Neurospora.

Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide.

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. Here we report the characterization of the photoinducible promoter of the albino-3 (al-3) gene, encoding GGPP synthase. We have modified the 5' non-coding sequence of the cloned al-3 gene by deletion and site-directed mutagenesis, and we have tested the residual photoinducibility of the different constructs by transformation and subsequent analysis of gene expression in dark-grown and light-induced mycelia. The results indicate that a promoter region between positions -226 and -55 contains all the necessary information for blue light photoinduction. Multiple regulatory elements are involved in the regulated expression of the al-3 gene. One (termed the APE element) is important for the light-induction switch on of the gene and a second belongs to the CCAAT boxes family. The putative APE element is also found in the promoter of other N. crassa photoinducible genes.

Suppression of gene expression by homologous transgenes.

When a wild-type strain of Neurospora crassa is transformed with different portions of the carotenogenic albino 1 or albino 3 genes, up to 30-35% of the transformants show an albino phenotype. The albino transformants presented a variety of phenotypes ranging from white or yellow to dark yellow colour. The ectopically integrated sequences provoke a severe impairment of the expression of the endogenous al-1 or al-3 genes. This phenomenon, that has been termed quelling, is found to be spontaneously and progressively reversible. In fact, all of the albino transformants have an unstable phenotype and revert progressively to wild type or intermediate phenotypes over a prolonged culturing time. The phenotypic reversion is characterised by a progressive release of the transcriptional inhibition and seems to correlate with the reduction of the number of the ectopic integrated sequences. However, there is no strict correlation between the copy number of the ectopic sequences and the intensity of quelling, as indicated by the existence of albino transformants containing only 1-2 ectopic sequences. The nature of the molecular events determining the onset of quelling is unclear, in any event, these are likely to involve some kind of interaction between the resident genes and ectopically integrated exogenous sequences. Recent evidences on a possible mechanism are presented.

Effect of two-week treatment with enprostil (35 micrograms twice a day) on 24-hour serum gastrin levels.

After a meal, a single dose of enprostil, a synthetic dehydroprostaglandin E2, inhibits gastrin level in both normal subjects and patients with duodenal ulcer, whereas H2 blockers exaggerate the postprandial gastrin response. However, the effect of prolonged treatment with enprostil on the gastrin profile is unknown. The aim of this study was to compare serum gastrin levels over a 24-hr period before (day 0) and on the last day (day 14) of a two-week course of enprostil (35 micrograms twice a day). Nine healthy volunteers (four women and five men), ages 29 +/- 5 years (range 23-39) were studied twice during a 24-hr period. Serum gastrin was measured at 30-min intervals during the day and at 2-hr intervals during the night. Enprostil (35 micrograms) was taken after basal gastrin serum measurement at 8:00 AM and PM. Standardized meals were ingested at 8:30 AM, 12:30 PM, and 8:30 PM. The postprandial integrated serum gastrin response was calculated after the three meals (4-hr period). Fasting serum gastrin levels were similar for the two periods. Integrated postprandial gastrin response was significantly inhibited after breakfast and dinner (P less than 0.001). Average results are expressed as mean +/- SEM (pmol/min/liter). During the night, gastrin levels were significantly decreased by enprostil. After 14 days, the inhibition of gastric acid secretion, which induces an increase of gastrin release with other antisecretory drugs, remained counterbalanced by the antigastrin properties of enprostil.