Genesilencing Phenomena 941 Silencing by Mutation RIP

Selker et al. (1987) investigated the underlying reason for the non-inheritance of transgenes in Neurospora. Specifically, they determined the fate of transforming DNA carrying single and duplicated DNA sequences and sequences that are normally unmethylated or methylated (Figure 9.3). A vector was constructed that had a single copy of the am+ gene and

Chromosomal region

Flank

Tandem duplication of "flank" DNA sequence by transformation

Flank am pUCB Flank

Figure 9.3 Selker et al.'s (1987) method of generating linked duplication of DNA sequence in N. crassa by homologous recombination of a plasmid. The only homologous sequence between pES174 and host was a 6 kb "flank" region (indicated by heavy line). The fate of this duplicated sequence between fertilization and nuclear fusion was studied.

a duplicated am+ flank region. This plasmid vector containing the wild type (am+) was used to transform an am (glutamate dehydrogenase) deletion mutant of Neurospora crassa to study the fate of a single copy of the am+ gene and a duplicated am+ flank region. The transformant was crossed to a strain lacking the am gene and the ascospore progeny were analyzed by Southern hybridization using the am gene probe to determine if they had inherited the transgene. The progeny showed a 10 kb band (Figure 9.4) that was not present in the primary transformant. This novel band could result only if the BamHI restriction sites b, c and d in the duplicated region had become modified. (BamHI is a restriction enzyme obtained from Bacillus amyloliquifaciens; it differs, for example, from EcoRI, a restriction enzyme from Escherichia coli.) By using a pair of restriction enzymes that distinguish methylated and unmethylated DNA (isoschizomers), the cut DNA upon elec-trophoresis shows a new band due to a change in the restriction sites by the modification of the cytosine residues in the DNA sequence by methylation of only the duplicated am flank region. The process that altered the extra (foreign) DNA sequences by mutations and epigenetic modification of the cytosine residues in DNA by methylation was named RIP, an acronym for repeat-induced point mutations. By dissecting out and analyzing ascospores meiotic tetrads (8-spored progeny asci), the timing of RIP could be determined by analyzing the meiotic tetrad. It was inferred that RIP detects and mutates duplicated sequences during pairing of homologous chromosomes prior to karyogamy in the ascus initial. RIP depends upon the capacity of premeiotic cells to recognize the presence of duplicated sequences in an otherwise haploid genome (Figure 9.5). The only copies of flank flank c d

2.3 kb 3.1 kb 4.7 kb 10 kb x Modified Bam H I site f g Bam H I sites

Fragments detected in Southern hybridization am

Transformat

Progeny Host am- am+

-10 kb^— (novel band)

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