Kenneth D. Tartof

Increasing the Multiplicity of Ribosomal RNA Genes in Drosophila melanogaster

In wild-type Drosophila melanogaster females there are about 250 ribosomal RNA genes in each nucleolus organizer region of the two X chromosomes. When this same nucleolus organizer region is present in flies in only a single dose, the number of ribosomal RNA genes increases to approximately 400. This increase is most easily explained by disproportionate replication of these genes.

A structural basis for variegating position effects

Variegating position effects in Drosophila result from chromosome rearrangements where normal genes, having been placed next to heterochromatin, are inactivated in some cells but not in others, thereby producing a variegated tissue. We have determined that the euchromatic breakpoints for three variegating white mutants are clustered and lie approximately 25 kb downstream of the white structural gene. In each case the white locus is adjoined in the heterochromatin to a mobile genetic element. Satellite sequences are not involved. We also demonstrate that revertants of the variegating mutant, wm4, are reinversions that leave the initial wm4-heterochromatic junction intact so that some heterochromatin-derived sequences remain joined to white at its new location. These results suggest a simple model for understanding the structure of heterochromatic domains and how variegating position effects may arise.

The 5 s RNA genes of Drosophila melanogaster.

Abstract 5 s RNA has been isolated from Drosophila melanogaster larvae. When submitted to electrophoresis on polyacrylamide gels, it migrates as a single sharp band, slightly ahead of mouse L-cell ribosomal 5 s RNA. It has a relatively high proportion (~52%) of guanylic plus cytidylic acid as compared to that of other ribosomal RNA species (38 and 41% for the 28 and 18 s components, respectively). DNA-RNA hybridization studies indicate that in wild-type males, females, or a mixed population of each, approximately 0.0065 to 0.0077% of the DNA is complementary to 5 s RNA, allowing an estimate of 195 to 230 genes for 5 s RNA per haploid genome. In the same stock approximately 0.31% of the DNA is complementary to 18 and 28 s ribosomal RNA indicating that there are at least 180 genes per haploid genome coding for these RNAs. Thus, it appears that in Drosophila the multiplicity for the various ribosomal RNA genes is roughly the same. To determine whether the 5 s genes are linked with the 28 + 18 s RNA genes, which are known to be on the sex chromosomes, flies containing appropriate deletions or duplications in the X chromosome were utilized. In these cases the number of genes coding for 5 s RNA was not detectably different from that in wild-type flies. It was therefore concluded that few, if any, of the 5 s genes reside in the X or Y chromosomes, and therefore that most, if not all, of the genes for 5 s RNA are located in the autosomes.

X and Y chromosomal ribosomal DNA of Drosophila: comparison of spacers and insertions.

Abstract In Drosophila melanogaster, the genes coding for 18S and 28S ribosomal RNA (rDNA) are clustered at one locus each on the X and the Y chromosomes. We have compared the structure of rDNA at the two loci. The 18S and 28S rRNAs coded by the X and Y chromosomes are very similar and probably identical (Maden and Tartof, 1974). In D. melanogaster, many rDNA repeating units are interrupted in the 28S RNA sequence by a DNA region called the insertion. There are at least two sequence types of insertions. Type 1 insertions include the most abundant 5 kilobase (kb) class and homologous small (0.5 and 1 kb) insertions. Most insertions between 1.5 and 4 kb have no homology to the 5 kb class and are identified as type 2 insertions. In X rDNA, about 49% of all rDNA repeats have type 1 insertions, and another 16% have type 2 insertions. On the Y chromosome, only 16% of all rDNA repeats are interrupted, and most if not all insertions are of type 2. rDNA fragments derived from the X and Y chromosomes have been cloned in E. coli. The homology between the nontranscribed spacers in X and Y rDNA was studied with cloned fragments. Stable heteroduplexes were found which showed that these regions on the two chromosomes are very similar. The evolution of rDNA in D. melanogaster might involve genetic exchange between the X and Y chromosomal clusters with restrictions on the movement of type 1 insertions to the Y chromosome.

Trans-sensing effects from Drosophila to humans

In drosophila and other dipterian insects ,homologous pairing of chromosomes in mitotic cells is a well established phenomenon.Of particular interest here are the significance of homologous pairing for gene expression in drosophila and its wider implications for mammals .

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.

Evolution of transcribed and spacer sequences in the ribosomal RNA genes of drosophila

Examination of the ribosomal RNA (rRNA) gene of six sibling species that make up the D. melanogaster subgroup reveals that the nontranscribed spacer is highly conserved during evolution. Indeed, the spacer is at least as conserved as the transcribed rRNA sequence in four of the six species and only slightly less conserved in the others. These data support the hypothesis previously suggested (Tartof and Dawid, 1976) that selection has a significant role in maintaining the parallel evolution of genetically separate but homologous redundant gene clusters.

Loss of heterozygosity studies indicate that chromosome arm 1p harbors a tumor suppressor gene for renal oncocytomas

We carried out a complete genome scan for loss of heterozygosity (LOH) in four renal oncocytomas by using highly polymorphic CA repeat microsatellite loci. Three of the four tumors exhibited LOH for chromosome arm 1p, and the oncocytomas of both female patients lost Xq. Therefore, these chromosome arms may harbor tumor suppressor genes involved in the etiology of this disease. Although the genomes of oncocytomas are relatively stable, two different microsatellite loci in one tumor were mutated by ±2 nt. Similar alterations in CA repeats that are probably due to spontaneous mutation have been observed in renal cell carcinomas. Genes Chromosom Cancer 16:64–67 (1996).

Restriction map of 5S RNA genes of Drosophila melanogaster

ALTHOUGH certain segments of the primary sequence and secondary structure of 5S RNA seem to be strikingly similar in prokaryotes and eukaryotes1, the genetic organisation of the 5S RNA genes themselves exhibits remarkable variability. In E. coli, several lines of evidence indicate that the multiple copies of the 5S, 16S and 23S rRNA genes are organised into transcription units containing one gene each plus a sequence coding either for tRNA1ILE or tRNA2GLU such that they are cotranscribed in the order 16S–4S–23S–5S RNA (refs 2 and 3). In eukaryotes, the 5S RNA genes may be clustered at a single site as in humans4,5 or widely distributed on many chromosomes as in the case of Xenopus laevis, where the 5S genes are found on the telomeres of most, if not all, of the 18 chromosomes6. Among the eukaryotes, there is no example in which the 5S genes are tightly linked to the 18S and 28S rRNA genes. Although there has been a large number of papers reporting the cytogenetic localisation of the 5S RNA genes in various organisms, only in the case of Xenopus laevis are there data concerning the physical organisation of these genes7. Even in this instance, however, the structure is only known for a few 5S gene repeat lengths, and the long range order that might be imposed on an entire 5S DNA cluster within the chromosome remains obscure. Whereas long range order in certain satellite sequences has been described8,9, similar higher order organisation has not as yet been revealed for transcribed redundant genes.

Molecular analysis of cubitus interruptus (ci) mutations suggests an explanation for the unusual ci position effects

The cubitus interruptus (ci) locus of Drosophila melanogaster is located proximally on chromosome 4. In ci mutants cubital wing veins are interrupted or absent. We have cloned this locus using a gypsy element associated with the ci1 mutation. Analysis of all extant ci mutations reveals that they contain conspicuous molecular alterations within a 13.7 kb region. Of the four homozygous viable mutations, three (ci1, ci36l, ciW) have single insertions, while one (ci57g) has a small deletion, all located within a more restricted 1 kb region. The dominant mutations, ciD and Ce2 each contain two insertions within the 13.7 kb region. All these molecular alterations are located upstream of a transcript previously associated with the ciD mutation and thought to derive from a segment polarity gene. We induced revertants of the dominant ci phenotype (wing vein interruption) in ciD and found molecular alterations in this transcript (the ci+ transcript) in two revertant alleles, thereby demonstrating this transcripts involvement in the ci phenotype. The locations of the molecular alterations, together with the results of the ciD reversion experiment, provide a connection between the dominant and recessive ci mutations and argue that all are likely to be alleles of the same complex locus, ci, not two separate loci as previously proposed. The ci phenotype of dominant and recessive mutations can be explained by inappropriate expression of the ci+ transcript in the posterior wing compartment where the cubital vein is affected, while loss of ci+ function generates recessive lethality. Lack of repression of ci+ transcription, through a pairing-dependent, trans-acting silencer element, can explain the unusual position effects associated with ci (the Dubinin effect).