Mild RIP-an alternative method for in vivo mutagenesis of the albino-3 gene in Neurospora crassa.
We have used a biological phenomenon that occurs in Neurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of the albino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-induced al-3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid bio-synthesis. Nucleotide sequence analysis of the al-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutated al-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes in N. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles of Neurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essential Neurospora genes.
Barbato
C
C
Dipartimento di Biopatologia Umana, Università di Roma, La Sapienza, Italy.
Calissano
M
M
Pickford
A
A
Romano
N
N
Sandmann
G
G
Macino
G
G
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D002338:Carotenoids / Q000096:biosynthesis; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005821:Genetic Techniques; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009492:Neurospora crassa / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D010641:Phenotype; D017354:Point Mutation; D012333:RNA, Messenger / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D012333:RNA, Messenger; D002338:Carotenoids; D010088:Oxidoreductases; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase