1

P-Carotene (pigment)

Figure 9.8 Carotenoid biosynthesis pathways showing the steps blocked in albino mutants.

albino genes of Neurospora provide a visual reporter system for identifying the silenced strains that have an albino phenotype (Romano and Macino, 1992).

The phenomenon of silencing of the endogenous (resident) and the transgene copies of genes in the vegetative phase was termed quelling. Transformation of the wild Neurospora strain with different portions of the al-1 gene shows that a minimum of about 130 bp of coding region can induce quelling. As this size would be insufficient to code for a functional protein, the requirement of transgene protein for quelling was ruled out. If the transforming DNA was al-3, Northern blot analysis shows that al-3 mRNA is absent, not al-1 or al-2 mRNA. The transcriptional silencing of homologous endogenous genes is the hallmark of gene-silencing events in fungi or plants. But whereas in plants almost all cases of gene silencing are associated with the repression of transcription due to the methylation of cytosine residues in the resident as well as the transgenes, quelling in Neurospora does not depend on methylation. This conclusion is based on results of Southern blot experiments using pairs of methylation-sensitive restriction enzymes, using an inhibitor of methylation or a mutant defective in methylation. To distinguish whether quelling is due to transcriptional inactivation or a post-transcriptional process such as RNA turnover, an RNAse protection assay was done. A labelled RNA probe complimentary to the al-1 gene was prepared and incubated with cellular RNA. After the unhybridized portion was removed by nuclease digestion, the size of the protected fragments was analyzed by gel electrophoresis. The amount of primary transcript (precursor mRNA) in quelled transformants was unchanged but the level of specific mRNA for the duplicated gene was reduced, leading to the conclusion that quelling is due to post-transcriptional gene silencing.

To understand the components of the machinery by which post-transcriptional silencing is brought about, a quelled strain (albino) was mutagenized and mutants (orange phenotype) were isolated that were impaired in quelling (Cogoni and Macino, 1997). By transformation of an albino-quelled strain with a plasmid (insertional mutagenesis), quelling deficient (qde) mutants were isolated that were orange in color. The rescued plasmid contained the putative qde gene whose sequence showed homology to RNA-dependent RNA polymerase. The experiments identified qde genes as a component of the silencing machinery. It is postulated that the qde product is RNA-dependent RNA polymerase that makes an antisense mRNA that causes the loss of transformed phenotype.

An interesting question arose whether the presence of transgene and endogenous gene in the same nucleus is required for silencing. A heterokaryon was constructed between quelled (albino phenotype) and wild strains (orange color) containing both al-1 silenced and non-silenced nuclei. The white color of the heterokaryon demonstrated that quelling is dominant and the presence of transgene and endogenous gene in the same nucleus is not a prerequisite for silencing. Moreover, the use of heterokaryon demonstrated that silencing could occur through a diffusible trans-acting molecule (Figure 9.9). A heterokaryon constructed between a qde mutant that produced no trans-genic sense RNA and a wild strain had an orange color, demonstrating that transgenic sense RNA is essential for silencing in heterokaryons. Presumably, the qde products and transgenic RNA interact to form a complex for degradation of endogenous mRNA and cause silencing.

Quelled, albino

Quelled, albino

Quelling-deficient, no transgene RNA, orange,

Wild type, orange

Wild type, orange

Quelling-deficient, orange Wild type, orange

Quelling-deficient, no transgene RNA, orange,

Wild type, orange

Wild type, orange

Figure 9.9 Diagram of quelling in heterokaryon. Gene introduced by transformation are shown in parentheses. Only one nucleus of each type is shown.

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Heterokaryon, albino

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Heterokaryon, albino

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