SCORING ABERRANT 4' : 4- ASCI IN SORDARIA BREVICOLLIS

: Frequencies of aberrant 4' : 4- asci are useful in checking on predic- tions of recombination models. In Sordaria brevicollis counting of aberrant 4' : 4-s is complicated by the occurrence of third division spindle overlap. A method is indicated where this snag could be overcome. It involves counting particular sequences of aberrant 4' : 4-s that are not masked by third division spindle overlap and then introducing a correction to obtain the true frequency of aberraht 4' : 4-s.

Aberrant 4+ :4-octads form a fairly crucial aberrant ascus class important in studies into the mechanism of genetic recombination. An aberrant 4+ : 4-that is produced by asymmetric hybrid DNA would require two independent recombinatibn events, namely a 5' : 3' event and a 3+ : 5event at corresponding sites.2 Hence by and large, aberrant 4' : 4's are good evidence for the occurence of symmetrical hybrid DNA. This fact was made use of by Paquette & Rossig-no17 to test a prediction of the Meselson -Radding model that asymmetric hybrid DNA was more frequent closer to the site of recombinant initiation.
Studies in aberrant 4+ : 4-s ought to be attempted mainly with spore mutants, for obvious reasons. Of all the fungal organisms that are normally used in genetic recombination studies, the only one in which aberrant 4+ : 4 s can be

is S o d a r i a f i m i c~l a .~
In Ascobolus immemus, where octads are unordered, aberrant 4+ : 4 s cannot be detected in a system having only a single spore marker. Ghikas & ~a r n b~ used a system involving two spore markers, a white (w) ascospore colour mutant and a granular (gr) ascospore mutant to detect aberrant 4+ : 4-s, while paquette6 used systems involving both tivo spore markers (white ascospore colour and round asgospore shape ; white ascospore colour and granular spore) and three spore markers (white spore colour, round spore shape and granular spore) to detect large numbers of aberrant 4+ : 4-s.
In Sordaria brevicollis the octads are relatively well ordered but the detection of aberrant 4+ : 4-s is complicated by the occurrence of third division spindle overlap. In this organism third division spindle overlap occurs to the extent of 2% -5%' whereas aberrant 4+ : 4-s may be at least two orders of magnitude lower in frequency.
Closer inspection shows that this snag of the occurrence of third division spindle overlap could be overcome. In an ascomycete when segregation of a marker is studied in asci, one can normally distinguish three classes of spore arrangementss in non-aberrant asci, namely the 4 : 4,2:2:2:2 and 2:4:2. The frequency of each of these three classes will be the nett result of the percentage of second division segregation and second division spindle overlap, but what is relevant to the present discussion are the frequencies of the three 'spore arrangements'. Each of the spore sequences shown in Figure 4 could belong to either of two spore arrangements, e.g. spore sequence 1 could be a 2:2:2:2 or a 2:4:2 depending on whether the chromatid that was initially wild type homoduplex and later became hybird, occupied the topmost position in the sequence or the second from top position in the sequence, respectively. Any ascus in any of the sequences 9 to 16 could similarly be 4:4 or a 2:4:2, while any ascus in any of the sequences 17 to 24 could be a 4:4 or a 2:2:2:2.
The position in the asci of the two chomatids, tbat were initially homoduplex and later became hybrid, will be eventually determined by both second division segregation and second division spindle overlap. Hence e-g. in the sequences 9-16 the ratio of asci showing the 4:4 spore arrangement to those asci showing the 2:4:2 spore arrangement will be the same whether one considered the total populaton of aberrant 4+ : 4-asci not masked by third divison spindle overlap and obtain the true frequency of aberrant 4" : 4's by introducing a correction. Let total frequency of sequences 9 -16 = x Then frequency of sequences 9 -16 that belong to the 4:4 spore arrangement = x (1-r-s). and the frequency of sequences 9 -16 that belong to the 2:4:2 spore arrangement = xs.
These will belong to the 4:4 and 2:4:2 arrangements in the ratio 1-r-s : s Therefore, the proportion of sequences 9 -16 that belong to the 4:4 Therefore the frequency of aberrant 4' : 4% that belong to the sequences 9-16 and belong to the spore arrangement Therefore from equation (2) and (3)  Therefore from equation (1) and (4) Since x, q, r and s are known, t, the total frequency of aberrant 4+ : 4 s is known.
Hence by counting the sequences 9 to 20 in figure 4 and using the frequencies of the 2:2:2:2 and 2:4:2 spore arrangements in the total sample of asci, the true frequency of aberrant 4+ : 4-s can be evaluated.
~h r e e of the sites s2,, S214 and s15, in theylo-9 spore colour locus in Sordaria brevicollis s t u d i e~i a v e rather low frequencies (3.1/104, 1.3/104 and 0.4110~ respectively, raw counts; these were made before the above method for correcting for the true frequency was worked out) of aberrant 4' : 4 s at 25OC. It is possible that other spore colour mutant sites give higher frequencies and that this method of obtaining the true frequencies of aberrant 4+ : 4-s in S. brevicollis may prove useful in obtaining data for checking predictions of recombination models.