University of Minnesota

The distribution of chromosome breaks in radiation hybrid lines from chromosome 3

--Okagaki, RJ, Jacobs, M, Stec, AO, Kynast, RG, Zaia, H, Granath, SR, Rines, HW, Phillips, RL

The distribution of breaks induced by radiation is believed to be randomly distributed along a DNA molecule. Therefore the number of breaks in an interval is dependent on the size of that interval. This principle forms the basis for radiation hybrid (RH) mapping and has been supported by work in a number of animal systems. Our lab has been engaged in developing RH lines derived from oat × maize chromosome addition lines. RH mapping panels for maize chromosomes 1, 2, 3, 4, 6 and 9 have been produced from the RH lines. Chromosome breaks in these lines were distributed reasonably evenly across the chromosomes, except for chromosome 3 RH lines. Over two-thirds (15/22) of the chromosome 3 RH lines have a break between markers umc1223 and mmp9 (data not shown). These markers are located at positions 234.4 and 262.9 cM on the chromosome 3 IBM2 genetic map; this distance is approximately 3.5% of the total genetic length of this chromosome. Because the probability of a radiation-induced break is proportional to the physical size of a region, this result suggests that a large proportion of chromosome 3 sequence resides in this small genetic interval. We are beginning to analyze this region first by correlating this region with the cytological map, and second by examining the sequence composition of the region.

Locations of breaks in RH lines were mapped relative to two translocations on chromosome 3. The translocation breakpoints in TB-3La and TB-3Sb have previously been placed on the Pioneer 1999 consensus map. This provided us with information that helped us develop markers to map the breakpoints on our RH lines. Five loci were selected that spanned a genetic interval where the two translocation breakpoints were expected to lie. These loci and their map positions were mmp69 (215.60), mmp29 (228.50), AY110403 (238.00), AY110297 (244.70), and AY110151 (254.60). Invader Assays were developed for these loci by Third Wave Technologies Inc. Invader Assays are quantitative assays that can be used to determine the copy number of a sequence. We have previously evaluated this assay by determining the copy number of individual r1 transcription units in a series of R-mb derivative alleles that have one, two or three copies of the transcription unit (R. Okagaki, unpublished data). A sequence that was on the translocated portion of 3L would be present in three copies in line TB-3La and in two copies in line TB-3Sb. A sequence that was present in two copies in lines TB-3La and TB-3Sb would not be present on either translocated arm. AY110403 and AY110297 were present in two copies in both TB-3La and TB-3Sb; therefore these two markers located at map positions 238.00 and 244.70 lay between the two translocation breakpoints. mmp29 at map position 228.50 was present in three copies in TB-3Sb and two copies in TB-3La. This placed the breakpoint in TB-3Sb between mmp29 and AY110403. Three copies of AY110151 were detected in TB-La and two copies in TB-3Sb. The breakpoint in TB-3La lay between AY110297 and AY110151. Data analysis for marker mmp69 has not been completed.

Markers mmp29, AY110403, AY110297, and AY110151 were then mapped on the RH lines. Assays were performed on up to 14 RH lines to determine the presence versus absence of each marker in a RH line. The bottom part of Figure 1 gives the mapping data from RH lines. The current set of RH lines divides chromosome 3 into six segments. The TB-3Sb breakpoint is in segment 3, and the TB-3La breakpoint falls in segments 3, 4 or 5. The middle portion of Figure 1 shows the locations of markers on the IBM2 map. Positions for markers umc10a, umc1223 and umc1683 were inconsistent between the two maps. The discrepancy was small for umc1223 and umc1683. Markers with an asterisk have not been placed on the IBM2 map. The top portion of Figure 1 depicts the two TB translocations used in this work and chromosome 3. The arm ratio of chromosome 3 is 2.0, and the region between the two translocation breakpoints is 20 to 25% of the chromosome length. The combined data show that the majority of chromosome breaks in our material occur in a region bounded by, or close to, the region defined by TB-3Sb and TB-3La.

Data from RH lines and cytological work suggests that this region of chromosome 3 accounts for much of the sequence on the chromosome while contributing little to the length of the genetic map. It seems possible that this region is largely composed of repetitive sequences. We are currently investigating this question. One preliminary Southern blot experiment has been conducted to compare the amount of the retroelement Grande1 in different RH lines. The signal intensity from line 3.1-015.4-01, which carries the long arm plus a proximal segment of 3S, was approximately twice that of line 3.1-035.1-05, which carries only long arm sequences. There are control experiments that must be completed, and additional work with other repetitive sequences remains to be done. However, if the initial results are supported, then a large fraction of the repetitive sequences along chromosome 3 are clustered in one region of the chromosome.