Plant genome studies: restriction fragment length polymorphism and chromosome mapping information.

The detection of sequence variation with restriction fragment length polymorphisms is advancing our knowledge of plant genetics on several fronts. In the past year, there has been progress in genetic map construction, phylogeny studies, and the dissection of multigenic traits. In addition, new methods that are independent of restriction sites are being developed for polymorphism detection.     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

Characterization of the genome of Arabidopsis thaliana

The small crucifer Arabidopsis thaliana has many useful features as an experimental organism for the study of plant molecular biology. It has a four-week life-cycle, only five chromosomes and a genome size less than half that of Drosophila. To characterize the DNA sequence organization of this plant, we have randomly selected 50 recombinant lambda clones containing inserts with an average length of 12,800 base-pairs and analyzed their content of repetitive and unique DNA by various genome blot, restriction digestion and RNA blot procedures. The following conclusions can be drawn. The DNA represented in this random sample is composed predominantly of single-copy sequences. This presumably reflects the organization of the Arabidopsis genome as a whole and supports prior conclusions reached on the basis of kinetics of DNA reassociation. The DNA that encodes the ribosomal RNAs constitutes the only major class of cloned nuclear repetitive DNA. It consists of approximately 570 tandem copies of a heterogeneous 9900-base-pair repeat unit. There is an average of approximately 660 copies of the chloroplast genome per cell. Therefore, the chloroplast genome constitutes the major component of the repetitive sequences found in A. thaliana DNA made from whole plants. The inner cytosine residue in the sequence C-C-G-G is methylated more often than the outer in the tandem ribosomal DNA units, whereas very few differences in the methylation state of these two cytosine residues are detected in unique sequences.     

Adjacent chromosomal regions can evolve at very different rates: Evolution of theDrosophila 68C glue gene cluster

Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.     

A trans-acting regulatory product necessary for expression of the Drosophila melanogaster 68C glue gene cluster

The mutation I(1)npr-1 is located at cytological location 2B5 on the X chromosome in Drosophila melanogaster. We have found that this mutation causes absence of the normal product of the 2B5 locus and that it has the following phenotypes: the 68C glue puff on the third chromosome does not regress when mutant salivary glands are cultured in the presence of ecdysterone; the three 68C glue protein mRNAs are not synthesized; and a transformed Drosophila strain carrying both a normal resident 68C Sgs-3 gene and an introduced functional Sgs-3 gene with only a few kb of flanking sequences expresses neither Sgs-3 RNA if the I(1)npr-1 mutation is crossed into the stock. Thus the normal product of the I(1)npr11 gene is required for regression of the 68C puff, and the I(1)npr-1 gene product allows expression of the Sgs-3 gene by interacting, either directly or indirectly, with DNA sequences near this glue protein gene.