Diversification of exogenous genes in vivo in Neurospora.

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.

DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon.

Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats.

DNA from the centromere region of linkage group (LG) VII of Neurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical of Neurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (approximately 16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobile copia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcen elements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in the Drosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.

Epigenetic control of a transposon-inactivated gene in Neurospora is dependent on DNA methylation.

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.

The Neurospora transposon Tad is sensitive to repeat-induced point mutation (RIP).

RIP (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the Neurospora crassa life cycle. Nevertheless, an active LINE-like retrotransposon, Tad, was found in a N. crassa strain from Adiopodoumé. The possibility was tested that Tad might be resistant to RIP, or that the Adiopodoumé strain might be incompetent for RIP. Tad elements derived from the Adiopodoumé strain were found to be susceptible to RIP. In addition, strains lacking active Tad elements, including common laboratory strains and strains representing seven species of Neurospora, were found to have sequences closely related to Tad but with numerous mutations of the type resulting from RIP (G:C to A:T). Even the Adiopodoumé strain showed Tad-like elements with mutations characteristic of RIP. Results of crossing of an Adiopodoumé transformant with progeny of Adiopodoumé suggest that the Adiopodoumé strain is proficient at RIP. We conclude that Tad is an old transposable element that has been inactivated by RIP in most strains. Finding relics of RIP in both heterothallic and homothallic species of Neurospora implicates RIP across the genus.

A simple and efficient system for targeting DNA to the am locus of Neurospora crassa.

We have developed a system of recipient strains and donor plasmids that allows targeting of DNA sequences to the am locus of Neurospora crassa. A recipient strain was constructed that contains the 3' two-thirds of the bacterial Hygromycin B (Hy) phosphotransferase-encoding gene (hph) in single copy downstream from the am structural gene encoding NADP-specific glutamate dehydrogenase. Plasmids have been constructed that contain am, but with the 5' end of hph downstream from am. Resistance to Hy can occur during transformation only if recombination occurs within the region of overlap of the hph sequences from the transforming DNA and of the chromosomal hph sequences. A lacZ alpha gene with a multiple cloning site has been inserted into the region between the am and hph sequences to facilitate subcloning into the targeting vector. Additionally, unique sites at the junctions of the vector and targeting DNA allow easy removal of vector sequences prior to transformation. A second recipient strain has been constructed that has had the am structural gene deleted. This strain may be used to provide a double selection for homologous integration. Additionally, this strain facilitates replacement of am with heterologous constructs, including constructs that drive transcription of am with different promoters. The insertion of hph downstream from am has no apparent effect on am expression, and the resistance segregates as a Mendelian factor in crosses. Transformation efficiency is reduced two to three orders of magnitude, but interestingly, nearly all of the resistant strains have only a single copy of the transforming DNA.

Tad1-1, an active LINE-like element of Neurospora crassa.

Tad is a LINE-like retrotransposon of Neurospora crassa. The element was originally detected and cloned using the am gene as a transposon trap in hybrid strains derived from a cross of Adiopodoume (a wild collected strain) and a laboratory strain devoid of Tad elements. We report the cloning and sequencing of an active Tad element, Tad1-1, which is capable of independent transposition. Transposition was demonstrated by screening for transfer of the element from a donor nucleus that contained the Tad1-1 element as the only active Tad, into a naive nucleus within a forced heterokaryon. We also report here the sequence analysis of Tad1-1, and its comparison with the sequence of another active element, Tad3-2. These elements are approximately 7 kb in length. They contain two long open reading frames (ORFs) encoded on the strand of the same polarity as the full-length transcript. ORF1 encodes a putative protein of 486 amino acids. Homology to the first ORF of other LINE elements is confined to three cysteine-rich motifs, located near the carboxy-terminus, that are thought to be involved in binding nucleic acids. The second ORF is 1156 amino acids in length and shows homology to the reverse transcriptase domains of various retroviruses and retrotransposons. Tad1-1 and Tad3-2 differ in only ten positions over their whole length.

Recurrence of repeat-induced point mutation (RIP) in Neurospora crassa.

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.

Repeat-induced G-C to A-T mutations in Neurospora.

In the Neurospora genome duplicate sequences are detected and altered in the sexual phase. Both copies of duplicate genes are inactivated at high frequency, whether or not they are linked. Restriction sites change, and affected sequences typically become heavily methylated. To characterize the alterations of the DNA, duplicated sequences were isolated before and after one or more sexual cycles. DNA sequencing and heteroduplex analyses demonstrated that the process (termed RIP) produces exclusively G-C to A-T mutations. Changes occur principally at sites where adenine is 3' of the changed cytosine. A sequence duplicated at a distant site in the genome lost approximately 10 percent of its G-C pairs in one passage through a cross. A closely linked duplication of the same sequence that was passed twice through a cross lost about half of its G-C pairs. The results suggest a mechanism for the RIP process.

Rearrangement of duplicated DNA in specialized cells of Neurospora.

Introduction of DNA into Neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. Sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, Neurospora sequences deleted from the host genome, and foreign sequences. The sequences already represented in the host were rearranged at high frequency in a cross. In general, both elements of the duplication, that from the plasmid and that from the host, became rearranged, whether or not they were linked. Unique sequences were left unaltered. Cytosine residues in the rearranged sequences typically became methylated de novo. Results from tetrad analyses indicated that the rearrangements occur before meiosis, during a stage between fertilization and karyogamy. We suggest that this previously unrecognized genetic process, RIP (rearrangement induced premeiotically), may contribute diversity for evolution and also maintain the gross organization of the genome.