Recombination at his-3 in Neurospora Declines Exponentially With Distance from the Initiator, cog

Corresponding author: D. E. A. Catcheside, Flinders University, P.O. Box 2100, Adelaide 5001, South Australia., david.catcheside{at}flinders.edu.au (E-mail)

Communicating editor: M. S. SACHS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

By deletion of 1.8 kb of sequence between cog^L and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog^L and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog^L, a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His⁺ recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log₁₀ (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog^L in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.

MEIOTIC recombination occurs more often in some regions of eukaryotic chromosomes than in others (LICHTEN and GOLDMAN 1995 ). Regions in which recombination is more frequent are assumed to include recombination hotspots (HOLLIDAY 1968 ).

Recombination hotspots have been studied in most detail at the HIS4 (NICOLAS et al. 1989 ) and ARG4 (DETLOFF et al. 1992 ) loci of Saccharomyces cerevisiae, at the his-3 locus of Neurospora crassa (ANGEL et al. 1970 ; CATCHESIDE and ANGEL 1974 ; YEADON and CATCHESIDE 1995 , YEADON and CATCHESIDE 1998 , YEADON and CATCHESIDE 1999 ; YEADON et al. 2001 ), and at the ade6 locus of Schizosaccharomyces pombe, where generation of a mutation, M26, fortuitously resulted in hotspot activity (GUTZ 1971 ).

Interallelic recombination in Neurospora has been shown to be due primarily to gene conversion (MITCHELL 1955 ; CASE and GILES 1958 ; STADLER 1958 ; SUYAMA et al. 1959 ; BOWRING and CATCHESIDE 1996 ), the nonreciprocal transfer of DNA sequence information that leads to non-Mendelian segregation of the sequence (FOGEL and HURST 1967 ). Conversion events are initiated close to recombination hotspots (DE MASSY et al. 1995 ; LIU et al. 1995 ; XU and KLECKNER 1995 ), which are usually located at the 5' end of a coding sequence (CATCHESIDE and ANGEL 1974 ; NICOLAS et al. 1989 ; DETLOFF et al. 1992 ; WU and LICHTEN 1994 ; ZAHN-ZABAL et al. 1995 ; BOWRING and CATCHESIDE 1998 ; YEADON and CATCHESIDE 1998 , YEADON and CATCHESIDE 1999 ). Conversion tracts usually extend in both directions from the initiation site (SCHULTES and SZOSTAK 1990 ; MALONE et al. 1994 ; BOWRING and CATCHESIDE 1998 ; YEADON and CATCHESIDE 1998 ), although there are exceptions to bidirectionality (PORTER et al. 1993 ).

Lengths of meiotic conversion tracts have been measured in S. cerevisiae (JUDD and PETES 1988 ; BORTS and HABER 1989 ), S. pombe (GRIMM et al. 1994 ), Neurospora (BOWRING and CATCHESIDE 1998 ; YEADON and CATCHESIDE 1998 ), and Drosophila melanogaster (HILLIKER et al. 1994 ). However, a mathematical relationship between the number of nucleotides separating two sites in a gene and the chance of co-conversion has been described in only two species, D. melanogaster (HILLIKER et al. 1994 ) and S. pombe (GRIMM et al. 1994 ).

At ade6 of S. pombe, the frequency of co-conversion of a silent marker with a selected mutant site decreased exponentially with increasing distance from the mutation (GRIMM et al. 1994 ). The minimum average tract length was estimated at 1 kb. Similarly, co-conversion data at the rosy locus of Drosophila revealed an exponential relationship between the distance between two sites and the chance that both sites would experience conversion (HILLIKER et al. 1994 ), with an average length of 352 bp for unselected and 706 bp for selected tracts. HILLIKER et al. 1994 suggested that the probability that a conversion tract will be at least a particular length x (in nucleotides) is ^x, where is the probability at each nucleotide that a tract will continue. This relationship was shown to be a good fit to the co-conversion data and generated an estimate of = 0.99717, with a standard error of 0.00026.

Interallelic recombination at the his-3 locus of Neurospora is initiated by the recombination hotspot cog in the absence of the dominant allele of the trans-acting rec gene rec-2⁺ (ANGEL et al. 1970 ; CATCHESIDE and ANGEL 1974 ; YEADON and CATCHESIDE 1998 ). cog is located centromere-distal (ANGEL et al. 1970 ) of the 3' end of the his-3 locus (BOWRING and CATCHESIDE 1991 ). rec-2⁺ reduces recombination in his-3 30-fold below that observed in a rec-2 homozygote (ANGEL et al. 1970 ). Recombination at cog is turned off by rec-2⁺ (CATCHESIDE and ANGEL 1974 ) and any residual recombination events are initiated close to the 5' end of his-3 (YEADON and CATCHESIDE 1998 ).

The cog initiation site is >2.2 kb from the 3' end of his-3 (YEADON et al. 2001 ), probably at or close to the 10-bp sequence required for the high-frequency recombination phenotype of cog^L (YEADON and CATCHESIDE 1998 ), which is located near the 5' end of the lpl gene at the centromere-distal end of the cog region (YEADON and CATCHESIDE 1995 ; YEADON and CATCHESIDE 1999 ).

By deletion of 1.8 kb of sequence between cog^L and his-3 and replacement with sequences of different lengths, we generated a set of Neurospora strains in which the distance between cog^L and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the altered strains includes cog^L, the more active allele of cog, and rec-2, which in combination ensure a high frequency of recombination in the vicinity of cog (ANGEL et al. 1970 ). In addition, each is a his-3 mutant (K26 or K480). Since the manipulated strains that have not been outcrossed are, except for the modification, genetically identical to the transfection recipients, this eliminates other genetic influences (CATCHESIDE 1970 ) on recombination frequency. Since we know that most interallelic recombination in his-3 is initiated by cog (ANGEL et al. 1970 ; CATCHESIDE and ANGEL 1974 ; YEADON and CATCHESIDE 1998 ) at positions located centromere-distal of the site of our modification (YEADON et al. 2001 ), the distance travelled by a recombination event that yields a His⁺ recombinant depends on the number of nucleotides between his-3 and the presumptive initiation site within the cog region. Thus, by altering the distance between cog and his-3 we can investigate the relationship between the distance travelled by a recombination event and the resultant frequency of recombinant progeny. We can then assess whether the exponential relationship between distance separating markers and the chance of co-conversion in Drosophila (HILLIKER et al. 1994 ) and S. pombe (GRIMM et al. 1994 ) holds for heteroallelic recombination in Neurospora.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Culture methods and media:
Except that crosses were supplemented with 200 µg/ml histidine, 500 µg/ml alanine, 500 µg/ml arginine, 200 µg/ml adenine, and 400 µg/ml lysine as required, culture methods and media were as described by BOWRING and CATCHESIDE 1996 . Vegetative cultures were supplemented with 200 µg/ml histidine, 500 µg/ml alanine, 500 µg/ml arginine, 400 µg/ml adenosine, and 400 µg/ml lysine as required.

Molecular methods:
Restriction enzymes, Klenow, and T4 DNA ligase were supplied by New England Biolabs (Beverly, MA) and were used according to the manufacturer's instructions. Electrophoresis used TAE and 1% agarose. The polymerase used for PCR was RedHot supplied by Abgene. PCR amplification used a Perkin-Elmer (Norwalk, CT) 2400 thermal cycler. Each 50-µl reaction included 0.5 units RedHot polymerase (Abgene), 200 ng of each primer, 1.75 µM MgCl₂, and 0.8 mM dNTP mixture (equal concentration of each). Cycling conditions were 94° for 1 min, then 30 cycles of 94° for 15 sec, 52° for 30 sec, and 72° for 3 min, followed by 72° for 7 min, using a Perkin-Elmer GeneAmp 2400 thermal cycler. Primers used were NEWXF-GGCACATAGAGTAATGATAACG and NEWC8R-ACTACAAGCAAAGTCGGG.

Construction of plasmid vectors and strains:
p(1-16)X was made by insertion of a 197-bp synthetic oligonucleotide (1-16) into the SpeI site (RASMUSSEN et al. 2002 ) of pJY64 (YEADON et al. 2001 ) between his-3 and the arg-2-TK construct (SACHS et al. 1997 ). p(17-32)X was made by deletion of 4 bp at position 77 in the TK coding sequence in pJY65 (YEADON et al. 2001 ), followed by insertion of a slightly different 197-bp synthetic oligonucleotide (17-32) into the SpeI site. pJY64, pJY65, pDV2-B5, and pDV3-B5 were made as described previously (YEADON et al. 2001 ; RASMUSSEN et al. 2002 ).

The his-3 K458 strains T11630, T11631, and T11644 were made as described previously (YEADON et al. 2001 ).

Strains T11783 and T11785 (Table 1 and Table 2), in which 1.8 kb between his-3 and cog was deleted (Fig 1), were made by transfection of T11644 with pDV3 and of T11630 with pDV2, respectively. The distance between his-3 and cog in the resultant monokaryotic strain (YEADON et al. 2001 ) was determined to be the same as that in the DV plasmids by electrophoresis of PCR products generated using the primers NEWXF and NEWC8R.

View larger version (42K): In this window In a new window Download PPT slide   Figure 1. Crosses in which we have altered the distance between K480 and cog. Light shading within his-3 indicates the sequence of Emerson a origin while dark shading indicates that of Lindegren Y8743 origin. Unmodified strains are depicted in the third diploid from the top. The centromere is to the left. Not to scale.

Strains T11738 and T11739 (Table 1 and Table 2), with a human immunoglobulin light chain gene inserted between his-3 and cog (Fig 1), were made as described previously (YEADON et al. 2001 ), by transfection of T11644 with pDV3-B5 and T11631 with pDV2-B5, respectively. Additionally, T11740, T11741, and T11742 were made by transfection of T11631 with pDV2-B5 (YEADON et al. 2001 ).

T11782 (Table 1 and Table 2) was made by transfection of T11644 with pDV2 (YEADON et al. 2001 ; RASMUSSEN et al. 2002 ). By electrophoresis of PCR products generated using the primers NEWXF and NEWC8R, the distance between his-3 and cog in the resultant monokaryotic strain (YEADON et al. 2001 ) was determined to be the same as that in T11644.

T11686 and T11690 (Table 1), strains with the arg-2-TK construct (SACHS et al. 1997 ) inserted between his-3 and cog (Fig 1), were made as described previously (YEADON et al. 2001 ). All other arg-2-TK strains (Table 1 and Table 2) were extracted from crosses between each of the primary transformants T11686 and T11690 and either T11668 or T11670 (Table 1).

Strains with the arg-2-TK construct (SACHS et al. 1997 ) and a 197-bp oligonucleotide inserted between his-3 and cog (Fig 1) were made in a similar way to T11686 and T11690 (YEADON et al. 2001 ). T11768 was made by transfection of p(1-16)X into T11630. T11769, T11770, and T11806 were made by transfection of p(17-32)X into T11644. Constructs were verified by Southern analysis (data not shown).

Strains T12002–T12004, with the egl3 constructs inserted between his-3 and cog (Fig 1), were supplied by Neugenesis (YEADON et al. 2001 ; RASMUSSEN et al. 2002 ).

Location of the position of the his-3 K480 mutant site:
The position of K480 was determined in a similar manner to those of K874, K26, and K1201 (YEADON and CATCHESIDE 1999 ), that is, by comparing the his-3 sequence from F10292 with that of the cosmid G3:6F (YEADON and CATCHESIDE 1999 ) made from the His⁺ strain ST74-OR23-IVA (ORBACH 1994 ).

Recombination assays:
Recombination frequency between his-3 alleles was measured by the method of ANGEL et al. 1970 . "Total A counts" (Table 2 and Table 3) reflects the number of His⁺ colonies generated by 75% of the spores from a cross. "Total C counts" (Table 2 and Table 3) reflects the number of colonies, both his-3 and His⁺, generated by 0.056% of the spores from a cross.

Calculation of distance (x bp) between K480 and the initiator in each strain:
Assuming that initiation occurs at the 10-bp sequence motif necessary for the high-frequency recombination phenotype of cog^L (YEADON and CATCHESIDE 1998 ), the initiation site is 3432 bp from the 3' end of the his-3 coding sequence in an untransformed cog^L strain. The his-3 sequence is 2676 bp long in total (YEADON and CATCHESIDE 1999 ), so that with knowledge of the position of K480, the value of x for an untransformed strain can be calculated. For each modified strain, x was determined arithmetically.

Statistical analysis of recombination data:
y, the log₁₀ of each recombination frequency (His⁺/10⁵ viable spores; Table 2), was plotted against x (Fig 2). The line of best fit was plotted, together with the equation describing the line and the r² value. This process was repeated (Fig 3) using only values of x for which the crosses involve only transformed strains that have not been outcrossed (1704, 2454, 4104, and 6054). The equation for the exponential relationship was calculated and used to predict the frequency of interallelic recombination at a theoretical distance of x = 0 and to predict the distance from cog at which the hotspot can no longer influence recombination within his-3.

View larger version (15K): In this window In a new window Download PPT slide   Figure 2. The relationship between distance from cog and recombination frequency at his-3 in all crosses. Recombination frequency is measured as histidine prototrophs per 105 viable spores. Columns of data points from left to right, respectively, correspond to the crosses depicted in Fig 1 from top to bottom.

View larger version (14K): In this window In a new window Download PPT slide   Figure 3. The effect of distance from cog on recombination in his-3 in diploids that differ only in the size of the his-3-to-cog interval. As in Fig 2, recombination frequency is measured as histidine prototrophs per 105 viable spores. However, in these crosses any influence of genes with small effects on recombination has been removed since diploids are genetically identical except for the modification between his-3 and cog.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Positions of his-3 mutant sites and distances between mutant pairs:
The K874 mutant site is at position +1717, K1201 is at position +115, and the K26 mutant site is at position +1502 (YEADON and CATCHESIDE 1999 ). The K480 mutant site is an A T transversion at +2605 in the his-3 coding sequence, resulting in a replacement of a lysine with a stop codon at codon 848. Thus the distance between K26 and K480 is 1103 bp and the distance between K1201 and K874 is 1602 bp. K874 is therefore 888 bp farther from the initiation site than is K480, so in K1201/K874 crosses the distance x is 4392 bp, 888 bp greater than the 3504 bp present in untransformed K480 strains (Fig 1).

The relationship between recombination and distance from the initiator:
Recombination frequencies (Table 2) increase exponentially with decrease in distance from cog (r² = 0.67; Fig 2). To obtain a better estimate of the effect that distance from cog has on recombination frequency in his-3, we considered which data sets might be influenced by factors other than this distance.

The untransformed strains (x = 3504 bp; Table 2) have different genetic backgrounds and it is likely that different alleles of genes with small effects on recombination (CATCHESIDE 1970 ) are present in each. In addition, T11681 and T11759, unlike all other strains used in this analysis, are cog^E (Table 1). Although the more active hotspot allele cog^L is ostensibly dominant to cog^E (ANGEL et al. 1970 ), the presence of cog^E in some of the crosses may contribute to the greater variability (Table 2 and Fig 2) in this data set (x = 3504 bp).

The TK⁺ strains (x = 3904 bp; Table 2) are all progeny of T11644 (Table 1), transformed with pJY64 or pJY65, and crossed to either T11668 or T11670 (Table 1). Thus alleles of genes with small effects on recombination (CATCHESIDE 1970 ) are likely to be segregating in the TK⁺ strains.

The data sets in which x = 1704, 4101, or 6054 bp (Table 2) are all from crosses between strains genetically identical to T11630 or T11644, except for the sequences within his-3 and those inserted into the interval between cog and his-3. The data set in which x = 2454 bp (Table 2) is similar, except that T11631, of the same parentage as T11630 and T11644, is substituted for T11630. Thus, in these crosses very little is variable except the distance x between cog and his-3, which has been altered deliberately.

Data from crosses in which the only difference is our modification between his-3 and cog (x = 1704, 2454, 4101, and 6054; Table 2) demonstrate clearly (Fig 3) that interallelic recombination increases exponentially as x decreases. The relationship is described by the equation log₁₀y = 2.9836–0.0002x [or y = 963(0.99954)^x] with r² = 0.928. Thus, when x = 0 (Fig 3) and K480 is adjacent to cog, y = 963/10⁵.

In the presence of rec-2⁺, which turns off initiation of recombination at cog^L (ANGEL et al. 1970 ), the frequency of His⁺ progeny from K26/K480 crosses is 1.5/10⁵ viable spores (data not shown). When y = 1.5 (Fig 3) and no detectable influence of recombination is initiated at cog^L, x = 14,038 bp.

Extrapolating from our data (Fig 3), we conclude that, if recombination were initiated adjacent to K480, the theoretical interallelic recombination frequency from a cross heteroallelic for his-3 K480/K26 would be 960 His⁺ progeny in 100,000 viable spores. In addition, if cog^L were 14 kb away from his-3, the effect of the hotspot would no longer be detectable at his-3, suggesting that the frequency of events initiated at cog^L that are longer than 14 kb is negligible.

Estimation of minimum frequency of recombination initiation at cog^L:
Because an initiation event in a single meiosis can result in two His⁺ and six His^- spores and because as calculated above it is theoretically possible to obtain 0.96% His⁺ progeny from a K26 x K480 cross, recombination must be initiated in at least 3.84% (4 x 0.96%) of meioses. This must be an underestimate, as it does not consider the chance that a recombination event may reach K480 without yielding a His⁺ spore.

Crosses heteroallelic for K874/K1201 yield His⁺ progeny at a frequency of 550/10⁵ viable spores (Table 3). The K874 mutant site is 888 bp farther from the initiation site than is K480, so for K874/K1201 heterozygotes, x = 4392 bp. Extrapolating from Fig 3 with an x value of 4392 bp, we predict 127 His⁺ progeny per 10⁵ viable spores. The recombination frequency in K874 x K1201 crosses (Table 3) is 4.3-fold higher than this estimate, probably reflecting the greater distance between K1201 and K874 (1602 bp, 599 bp more than the 1103 bp between K480 and K26). Since the yield of His⁺ progeny from crosses heteroallelic for K26/K874, in which only 214 bp separate the mutant sites, is substantially reduced (ANGEL et al. 1970 ) and, in our system, the choice of the his-3 allele is unlikely to influence initiation frequency, 3.84% must be an underestimate of the frequency of initiation of recombination in crosses homozygous for cog^L by at least a factor of 4.3. We therefore conclude that a more accurate estimate is that recombination is initiated at cog^L in 17% (3.84% x 4.3) of meioses. Assuming that distances between mutant sites greater than that separating K1201 and K874 would yield still greater numbers of His⁺ progeny, even this estimate is conservative.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The data presented here (Fig 3) show that recombination events initiated by cog^L are distributed exponentially with respect to distance travelled. Thus we have shown that the exponential relationship between the distance separating two sites and the chance of co-conversion described at the rosy locus of Drosophila (HILLIKER et al. 1994 ) and at ade6 of S. pombe (GRIMM et al. 1994 ) extends to the relationship between the his-3 heteroallelic recombination frequency and the distance from cog in Neurospora. For co-conversion, the probability that a conversion tract will be at least a particular length x (in nucleotides) is ^x, where is the probability at each nucleotide that a tract will continue (HILLIKER et al. 1994 ). The formula presented here, y = 963(0.99954)^x, which describes the relationship between interallelic recombination frequency at his-3 and the distance from the recombinator, is of a similar nature.

The formula derived for Drosophila describes the chance of co-conversion of a nucleotide site at a distance x from a site already showing conversion. When x is zero and the sites are adjacent, the chance of co-conversion combines (a value closely approximating unity) and the chance of conversion rather than restoration (a probability also close to one, as adjacent sites are likely to be covered by the same mismatch repair tract; DETLOFF and PETES 1992 ). So, the chance of co-conversion of adjacent sites is approximately equivalent to . In contrast, the data presented here do not reflect co-conversion but rather the relationship between interallelic recombination frequency at his-3 and distance from the recombinator. Thus, in addition to the probability that a conversion tract will continue (^x), we must also consider the probability of initiation per spore (i) and the chance (k) that a tract, after passing K480, will result in a His⁺ spore, resulting in an equation of the nature y = k.i.(^x), exactly the type of equation found to be a good fit for the data presented here. We suggest that both and the probability of initiation are constant at this locus in crosses homozygous for cog^L, but that k is variable and depends on the nature of each of the mutant sites and on the number of nucleotides separating the sites.

As yet, we cannot accurately estimate , as the effects of k and i cannot be separated from that of in this data set. However, co-conversion data collected previously during mapping of conversion tracts at his-3 (YEADON and CATCHESIDE 1998 ) suggest that for this locus of Neurospora is greater than the 0.99717 estimated for the rosy locus of Drosophila (HILLIKER et al. 1994 ). Of tracts in which his-3 K26 was converted, only 2 of 15 extended less than another 270 bp, 11 extended more than 270 and less than 939 bp, and an additional 2 extended between 939 and 1387 bp past K26 (YEADON and CATCHESIDE 1998 ). If were as low as 0.99717, only 46% of the tracts should have extended >270 bp past K26, significantly less than the number observed (² = 9.8; P < 0.01). Substituting 0.99954 (from the equation for y, shown above) for the Drosophila value of leads to the prediction that 87% of tracts would extend >270 bp past K26 and, in fact, 13 of 15 (87%) did so, suggesting that for this locus of Neurospora may be of this order.

However, when cog^L is >14 kb from his-3, the frequency of His⁺ recombinants is predicted to be similar to that obtained when recombination is turned off by rec-2⁺, suggesting that few events travel farther than 14 kb. The exponential relationship between distance from cog^L and the frequency of His⁺ recombinants obtained from a cross indicates that most events will travel only a relatively short distance. Also, in crosses homozygous for cog^L, we estimate that recombination is initiated by cog^L in at least 17% of meioses although, since this estimate does not consider the chance that a conversion event may pass a mutant site without yielding a His⁺ spore, the actual frequency of initiation is likely to be much greater.

	ACKNOWLEDGMENTS

We thank John Yeadon for his help with the mathematics of curves and Russell Stainer for his technical assistance. This work was supported by a grant from the Australian Research Council and by Flinders Technologies. Strains T12002 and T12004 and clones including genes were supplied by Neugenesis, San Carlos and Berlex Laboratories (Richmond, CA).

Manuscript received March 14, 2002; Accepted for publication July 8, 2002.

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