Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells.

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.

A heat-shock-activated cDNA encoding GAGA factor rescues some lethal mutations in the Drosophila melanogaster Trithorax-like gene.

GAGA factor is an important chromosomal protein involved in establishing specific nucleosome arrays and in regulating gene transcription in Drosophila melanogaster. We developed a transgenic system for controlled heat-shock-dependent overexpression of the GAGA factor 519 amino acid isoform (GAGA-519) in vivo. Efficient production of stable protein from these transgenes provided genetic rescue of a hypomorphic Trithorax-like (Trl) lethal allele to adulthood. Nevertheless, supplemental GAGA-519 did not suppress position effect variegation (PEV), a phenomenon commonly used to measure dosage effects of chromosomal proteins, nor did it rescue other lethal alleles of Trl. The results suggest requirements for the additional isoforms of GAGA factor, of for more precise regulation of synthesis, to carry out the diverse functions of this protein.

Chromatin organization and transcriptional control of gene expression in Drosophila.

It is increasingly clear that the packaging of DNA in nucleosome arrays serves not only to constrain the genome within the nucleus, but also to encode information concerning the activity state of the gene. Packaging limits the accessibility of many regulatory DNA sequence elements and is functionally significant in the control of transcription, replication, repair and recombination. Here, we review studies of the heat-shock genes, illustrating the formation of a specific nucleosome array at an activatable promoter, and describe present information on the roles of DNA-binding factors and energy-dependent chromatin remodeling machines in facilitating assembly of an appropriate structure. Epigenetic maintenance of the activity state within large domains appears to be a key mechanism in regulating homeotic genes during development; recent advances indicate that chromatin structural organization is a critical parameter. The ability to utilize genetic, biochemical and cytological approaches makes Drosophila an ideal organism for studies of the role of chromatin structure in the regulation of gene expression.

Mutations that affect ion channels change the sensitivity of Drosophila melanogaster to volatile anesthetics.

We have quantitated the response of D. melanogaster to general anesthetics with a device, the inebriometer, that assays the fly's geotactic and postural behavior. When alleles of several loci that encode or regulate subunits of ion channels were compared with control stocks, several ion channel mutants clearly increased the anesthetic sensitivity of Drosophila. The effects were specific in that: a) for several alleles, genetic tests indicated that the anesthesia phenotype was due to the ion channel mutation and not to extraneous genetic differences between the stocks; b) a given ion channel mutation often affected the response to one anesthetic but not another; and c) the behavior of decapitated flies in the inebriometer indicated that the anesthetic phenotype of several mutants did not merely reflect a global change in the fly's physiology. These results provide support for the idea that ion channels are on the pathway(s) influenced by anesthetics and that different anesthetics use different pathways. They also provide perspective on the behavior of previously isolated mutations (har) that decrease the sensitivity of Drosophila to anesthetics in the inebriometer.

Chromatin. Ga-ga over GAGA factor.

Recent results suggest that the Drosophila transcriptional activator known as GAGA factor functions by influencing chromatin structure.

Increase in the number of histone genes in case of their deficiency in Drosophila melanogaster.

We have determined the number of histone structural genes in D. melanogaster heterozygotes for two different deficiencies of a histone locus in the 2d chromosome. The results indicate a possibility of histone genes increasing in number in the case of their deficiency through magnification and compensation, as has been shown for rRNA genes by other authors.

Influence of deficiency of the histone gene-containing 38B-40 region on X-chromosome template activity and the white gene position effect variegation in Drosophila melanogaster.

The deficiency of the 38B-40 region containing histone genes in one of the 2nd chromosomes of D. melanogaster triploid intersexes increases the template activity of X-chromosomes both in vivo and in vitro without noticeably affecting autosome activity. This deficiency in the heterozygous state inhibits the variegated position effect of the white gene in the T(1;3)Wvco translocation in diploid females and males, but not affect their rate of development. The variegation suppressor Su(var)hg-1 not only suppress the gene position effect in diploid flies, but also increases the template activity of X-chromosomes in triploid intersexes. The results are discussed with respect to the dependence of gene activity on the structure of chromosomes (density of DNP packing).

Comparison of in vivo and in vitro RNA synthesis on polytene chromosomes of Drosophila.

A comparative radioautographic study of the RNA precursors incorporation on polytene chromosomes of Drosophila in vivo in the cells of salivary glands, and in vitro during incubation of E.coli RNA polymerase on slides with fixed chromosomes was performed.--The pattern of in vivo 3H-uridine incorporation on different sections of the chromosomes drastically differed from the in vitro 3H-UTP incorporation which seems to be much more related to DNA content of the individual small sections. In both cases puffing of the loci resulted in the increase of RNA synthesis but in vitro only 2-3 fold and in vivo much more. Hence, RNA synthesis in vitro was unspecific and did not reflect the in vivo RNA synthesis.--On the other hand, E.coli RNA polymerase completely mimics in vitro the dosage compensation phenomenon making twice as much RNA on one X-chromosome of males (1X2A) as on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females and super-females (3X2A), and the intermediate amount of RNA on each of X-chromosomes of intersexes (2X3A). It is suggested that the differences in the in vitro template activity of X-chromosomes of cells with different X:A ratio are due to different extent of condensation of their deoxyribonucleoprotein (DNP). Yet, both male and each of female X-chromosomes bind the same amount of thymus histone FI labelled with fluorochrome which indicates that they contain the same amount of "open" regions with exposed chromosomal DNA accessible to external proteins.--On the basis of these observations a hypothesis is put forward which suggests that RNA transcription in animal chromosomes is regulated at two levels by different mechanisms; the first one controls the extent of condensation of DNP of genetic loci and determines their competence to the second mechanism which involves the action of gene-specific activator proteins. According to this hypothesis the phenomenon of dosage compensation of sex-linked genes is due to decondensation of DNP of male X-chromosome which renders its loci twice as responsive to activators as compared to the same loci in females.