Twenty-three homozygous interchange stocks with a break in the short arm of chromosome 6 were used to study the distribution of rRNA genes in various segments of the nucleolus organizer region (NOR). Labeled rRNA was prepared either by iodination of 18S rRNA with 125I-rRNA or by cRNA transcribed from cloned maize rDNA using 3H-nucleoside triphosphates. In situ hybridization (Table 1) with 125I-rRNA and 3H-cRNA of interchanges T5-6d, T4-6(7328) and T2-6(001-15), all with a break in the chromosome 6 satellite (MGCNL 51:52), indicated that rRNA genes are not prevalent beyond the first chromomere of the satellite; no silver grains were observed over the second or third chromomere when translocated to other chromosomes. Interchange T1-6d has a break between the centromere and the NOR in the short arm of chromosome 6. Hybridization of T1-6d revealed the expected distribution of rRNA genes; all silver grains were localized over the NOR.
The cloned rDNA includes the non-transcribed spacer region (Benton, Ph.D. Thesis, Univ. Minn.). The 3H-cRNA hybridized only to the NOR of the 15 homozygous interchanges and the inbred line A188. This observation suggests that the spacer sequence is probably not present at genomic locations other than the NOR.
At least part of the NOR-secondary constriction was separated from the NOR heterochromatin in 11 homozygous interchanges, namely T1-6(8415), T3-6(030-8), T1-6(4986-7), T2-6(027-4), T5-6f, T1-6(5495), T4-6Li (previously listed as T1-6Li), T3-6(032-3), T2-6(5419), T6-7(035-3) and T5-6(8696) (in Maize Breeding and Genetics, edited by Walden, 1978). Silver grains were observed over both segments after hybridizing with labeled rRNA. This indicates that the NOR-secondary constriction contains rRNA genes.
The cytological placement of breakpoints agrees well with the hybridization data. Cytology placed the break of T5-6f midway in the secondary constriction. Hybridization data suggested the break was in the proximal portion. This minor discrepancy was probably due to the difficulty in assigning the exact cytological breakpoint.
Interchange T6-10(5519) has a break at .90 in the NOR-heterochromatin and in situ hybridization revealed 70% of the silver grains over the proximal 90% of the NOR-heterochromatin (P/(P+D) ratio of .70). Interchanges T1-6(8415) and T3-6(030-8) both have a break in the proximal segment of the NOR-secondary constriction and also gave a 70% P/(P+D) ratio. These results suggest that 70% of the rRNA genes are localized in the NOR-heterochromatin and 30% in the NOR-secondary constriction.
The conclusion that 70% of the rRNA genes reside in the NOR-heterochromatin is in contrast to the 90% estimate of Givens and Phillips (Chromosoma 57:103-117), obtained by filter DNA/rRNA saturation hybridization. The discrepancy may be due to differential accessibility of the DNA for in situ hybridization between the NOR-heterochromatin and NOR-secondary constriction. The NOR-secondary constriction may be more readily hybridized than the NOR-heterochromatin.
R. L. Phillips and A. S. Wang
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