Polytene chromosome hybridization

Young7 screened 80 randomly isolated plasmids from a genomic library of D. melanogaster DNA for repetitive elements. Of these 23 were found to be repetitive by hybridization to Southern blots of D. melanogaster DNA, and 17 of these were interspersed repetitive elements as shown by in situ hybridization with polytene chromosomes. 

This gene codes for an 0.8 kb mRNA and conceptual translation of the cochng region revealed a peptide that shows 69% identity to Manduca EH. In situ hybridization to the polytene chromosomes from larval salivary glands showed that the Drosophila EH gene is located on the right arm of chromosome 3 at location 90B. This region does not correspond to any known mutations, but stocks are available with deletions in this area to facilitate a screen for mutants at the EH locus. 

The highly reproducible banding patterns of polytene chromosomes make it possible to localize cloned Drosophila DNA on a Drosophila chromosome by in situ hybridization (Figure 10-30) and to visualize chromosomal deletions and rearrangements as changes in the normal pattern of bands. 

However, Spear and Gall (1973) questioned whether somatic magnification does occur in Drosophila. rRNA—DNA saturation hybridization experiments with DNA isolated from diploid tissues revealed that XX flies contain about twice as much rDNA than XO flies. Thus, in diploid cells, the rRNA genes are present in amounts proportional to the number of nucleolus organizers. In contrast to the situation in diploid cells, polytene chromosomes of the salivary glands from XX and XO flies, respectively, contain the same amount of rDNA. Since it is known that adult Drosophila flies do contain polytene chromosomes, the rDNA increase in XO flies reported by Tartof (1971, 1973) could result from the relatively higher amount of rDNA present in polytene chromosomes... 

The gene for Drm-SP-1 has been localized by in situ hybridization to polytene chromosomes at 70A (20). This 266 bp single-copy gene with one intron has been cloned and expressed when linked to the heat shock promoter or the yolk protein 1 promoter (E. Kubli and coworkers as cited by (41)). Other aspects of Drm-SP-1 molecular biology are summarized by Chen (41). 

The differences in the characteristics of DNA replication could play an important role in differential transcriptional activity of homologous chromosomes, especially in interspecific hybrids. It was demonstrated that in polytene chromosomes of D. melanogaster there is positive correlation between transcription intensity and the rate of replication of the corresponding X chromosomes (Berendes, 1966a,b Ananiev and Gvozdev, 1974 a, b). 

Differences in DNA syntheses in homologous regions of polytene chromosomes were also demonstrated for Anopheles atroparvus x A. labranchiae hybrids (Tiepolo et al, 1974). 

Heat the staining solution (but do not boil) and filter. If stored tightly capped, it will last for many years. The same solution, but without the orcein stain, can be used to prepare polytene chromosomes for in situ hybridization. 

N SITU HYBRIDIZATION TO POLYTENE CHROMOSOMES Can be used to answer several kinds of questions. For instance, the technique is frequently used to find the chromosomal site of a cloned DNA sequence, as well as to investigate the distribution of families of repeated sequences in the chromosome set and to measure the amount of a repeated sequence at different sites. These different experimental goals are best accomplished by variations of the technique. 

This protocol is a modification of the procedure described by Birnboim and Doly (1979). For most bacteriophage PI clones, the protocol yields a sufficient amount of DNA such that 15-20 ll can be used per restriction digest in a Southern blot. We typically use 1.5 pi per sHde for in situ hybridizations with polytene chromosomes. DNA yield strongly depends on the specific bacteriophage PI clone and may range from 3 to 15 pg. 

Preparation of polytene chromosomes for in situ hybridization Protocol 25... 

EXPERIMENTAL FIGURE 10-30 Banding on Drosophila polytene salivary gland chromosomes and in situ hybridization are used together to localize gene sequences. 

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