Multidrug resistance phenotype in patients with chronic lymphocytic leukemia as detected by immunofluorescence (FACS) and northern blot analysis.

The multidrug resistance (MDR) phenotype has been demonstrated to be related to the overexpression of P-glycoprotein, a 170 kDa transmembrane efflux pump. We studied P-glycoprotein expression in 40 patients with chronic B-cell leukemias by FACS analysis using MoAb c219, which recognizes both the MDR1 and the MDR3 gene product. We found significantly elevated P-glycoprotein expression in these patients as compared with normal controls. Patients who had received previous chemotherapy regimens containing MDR-related drugs showed significantly higher P-glycoprotein expression with MoAb c219 than those patients who had been untreated. Northern blot analysis of MDR1 and MDR3 gene expression in 32 of the patients gave a similar result: in the analysis of total RNA four of six patients (66%) pretreated with either vinca alkaloids or anthracyclines were MDR1 positive as opposed to 6 of 26 (23%) who had no treatment or treatment without these agents. In contrast, MDR3 expression was found more frequently (63%), but was randomly distributed in the differently treated groups. Increasing the sensitivity level by analysis of enriched mRNA (polyA+RNA) led to the detection of MDR1 and MDR3 expression all B-CLL patients. We conclude that a basic elevated P-glycoprotein expression is intrinsic in CLL cells, which is possibly upregulated under chemotherapy. This might be responsible for initial and acquired chemotherapy resistance in CLL patients. Follow-up of the B-CLL patients over 46 months showed that the median survival time for MDR1+ patients was 19 months as opposed to 46 months for MDR1- patients (p < 0.01). There was no statistical difference in survival between MDR3+ and MDR3- patients. In the MDR1+ group, eight of nine patients had developed resistance to the therapy with MDR-related drugs. The expression of MDR1 might, therefore, predict treatment failure with MDR-related drugs and be a negative prognostic factor.

Krüppel, a gene whose activity is required early in the zygotic genome for normal embryonic segmentation.

Embryos homozygous for Krüppel die as late embryos with an altered segmentation pattern. In strong alleles the normal thoracic and anterior abdominal segments are replaced by a partial mirror image duplication of the posterior abdomen. Weak alleles cause smaller pattern deletions in the thorax and abdomen and are not associated with mirror image duplications. The altered segmentation pattern can be traced back to 12 min after the onset of gastrulation, when the shorter germ bands in homozygous Kr embryos provide a first indication of abnormal patterning. The mutant was mapped to position 107.6 at the tip of the right arm of the second chromosome, cytologically to bands 60F2-5. Analysis of homozygous deficiency embryos indicate that the phenotype produced by strong point mutations probably represents the amorphic condition. The requirement for Kr+ gene activity is strictly zygotic. Maternal dosage of Kr+ has no effect on the embryonic phenotype, nor does homozygosity for Kr prevent germ cells from making normal eggs capable of normal embryonic development when fertilized by wild-type sperm. The requirement for Kr+ seems restricted to embryogenesis. Homozygous clones induced in imaginal discs during larval development survive and develop normally and in vivo cultures established from homozygous embryos proliferate normally and metamorphose into adult structures of normal morphology.

Balbiani ring induction in phosphate metabolism.

Balbiani rings (BR), giant puffs in Chironomus larval salivary glands, code for giant secretory proteins. As shown earlier, the normally dominant BR2 is turned off with its putative translation product during exposure of larvae to compounds that diminish the stores of P(i). A BR6 develops from a compact chromosome band, and a new giant protein appears in the secretion as the major component. We have determined the sequence of cloned DNA fragments representative for large parts of BR1 and BR2 (normally active) and the inducible BR6. There is an excess of positive charges and high contents of serine/threonine in the coded amino acid composition for the BR1 and BR2 sequences. The coded amino acid sequence for the BR6 clone shares homologies with the others but has an excess of negative charges and lacks serine/threonine. This suggested that the P(i) effects observed earlier could be related to differences in phosphorylation between the normal proteins and the BR6 product. This could be confirmed by measurements of phosphorylation, which occurs in the normal giant proteins mainly at seryl residues. P export with giant secretory protein is normally quantitatively important. Thus, BR6 activation should decrease P loss when P(i) pools are lowered because of inducer action.

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : I. Zygotic loci on the second chromosome.

In a search for embryonic lethal mutants on the second chromosome ofDrosophila melanogaster, 5764 balanced lines isogenic for an ethyl methane sulfonate (EMS)-treatedcn bw sp chromosome were established. Of these lines, 4217 carried one or more newly induced lethal mutations corresponding to a total of 7600 lethal hits. Eggs were collected from lethal-bearing lines and unhatched embryos from the lines in which 25% or more of the embryos did not hatch (2843 lines) were dechorionated, fixed, cleared and scored under the compound microscope for abnormalities of the larval cuticle. A total of 272 mutants were isolated with phenotypes unequivocally distinguishable from wild-type embryos on the basis of the cuticular pattern. In complementation tests performed between mutants with similar phenotype, 48 loci were identified by more than one allele, the average being 5.4 alleles per locus. Complementation of all other mutants was shown by 13 mutants. Members of the complementation groups were mapped by recombination analysis. No clustering of loci with similar phenotypes was apparent. From the distribution of the allele frequencies and the rate of discovery of new loci, it was estimated that the 61 loci represent the majority of embryonic lethal loci on the second chromosome yielding phenotypes recognizable in the larval cuticle.

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster : II. Zygotic loci on the third chromosome.

The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.

Constant and variable parts in the Balbiani ring 2 repeat unit and the translation termination region.

The large Balbiani rings of the chironomids produce giant internally repeated transcripts that are translated into silk-like proteins used for protective tubes. We have cloned fragments of the Balbiani ring 2 (BR2) gene of Chironomus pallidivittatus, normally the most prominent BR, for sequencing and restriction analysis. The results indicate a basic, tandemly arranged repeat unit of 198 bp, consisting of an invariant region of 102 bp followed by a variable region of 96 bp, the latter containing short internal tandem repeats. In the coding strand of both regions, there is a tendency to maximize adenine ( 40%) and minimize cytosine + thymidine (32-33%). Stringency in codon usage and absence of preference for third position nucleotide substitutions between homologous sequences suggest selection at the nucleotide level to be important. Both regions code for peptides rich in basic amino acids, but show distinct differences in other coding properties. The invariant region probably codes for a crystalline domain, and the variable region for a proline-rich, amorphous domain. One of the clones includes the 3' end of the translated region. Surrounding and following two stop codons is a sequence of four short palindromes. Furthermore, the first stop codon is part of a sequence reminiscent of a "TATA box". The possibilities are discussed that this area of the gene might be a target for regulatory molecules controlling translation termination and/or the expression of an overlapping cistron.

Initiation of lambda DNA replication in vitro promoted by isolated P-gene product.

The product of the P gene of bacteriophage lambda was isolated from heat-induced lambda-lysogenic Escherichia coli cells. It was found to bind to DNA, to be devoid of nuclease activity acting on double-stranded lambda DNA and of nicking/closing activity. Initiation of lambda DNA replication promoted by the P-gene product in a complementation assay in vitro was sensitive to rifampicin. Sedimentation analysis of the products and their hybridization to separated lambda DNA strands indicate that lambda DNA was formed in a reaction similar to ring-to-ring replication in vivo. The reaction was symmetric from the beginning, i.e. both lambda DNA strands were copied without delay.