Luchezar Karagyozov

A Single Amino Acid Exchange Inverts Susceptibility of Related Receptor Tyrosine Kinases for the ATP Site Inhibitor STI-571

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) α- and β-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit is, however, not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the β5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGFβ-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGFβ-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models of the PDGFβ-receptor and Flt3 kinase domains and comparison with the crystal structure of the STI-571 Abl kinase complex indicated very similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly motifs and activation loops. Accordingly, we observed reduced sensitivity of preactivated compared with nonactivated PDGFR-β for the inhibition by STI-571. Courses of the activation loop that collide with STI-571 binding explain its inactivity at other kinases as the insulin receptor. The binding site models of PDGFR-β and Flt3 were applied to predict structural approaches for more selective PDGFβ-receptor inhibitors.

Hereditary motor and sensory neuropathy - Lom (HMSNL): Refined genetic mapping in Romani (Gypsy) families from several European countries

Hereditary motor and sensory neuropathy type Lom, initially identified in Roma (Gypsy) families from Bulgaria, has been mapped to 8q24. Further refined mapping of the region has been undertaken on DNA from patients diagnosed across Europe. The refined map consists of 25 microsatellite markers over approximately 3 cM. In this collaborative study we have identified a number of historical recombinations resulting from the spread of the hereditary motor and sensory neuropathy type Lom gene through Europe with the migration and isolation of Gypsy groups. Recombination mapping and the minimal region of homozygosity reduced the original 3 cM hereditary motor and sensory neuropathy type Lom region to a critical interval of about 200 kb.

The structure of the 5′-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5′-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG356. Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG191, leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG191 and AUG356 inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5′-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation.

Effect of cycloheximide on the in vivo and in vitro synthesis of ribosomal RNA in rat liver

The action of low (5 mg/kg body wt;) and high (20 mg/kg body wt.) doses of cycloheximide, both causing a rapid and almost complete inhibition of protein synthesis in rat liver is investigated. Short-term (15 min) [14C]orotate incorporation into nucleolar rRNA in vivo is inhibited only by the high dose acting for periods longer than 1 h. The effect may be correlated with a strongly reduced labelling of the cellular pool of free uridine nucleotides. These results indicate that in vivo transcription of rRNA genes may not be under stringent control. The activity of template-bound RNA polymerase A in nuclei isolated from animals treated with both doses of cycloheximide is reduced within 1 h to about 50% of controls reaching nearly plateau levels at longer times of action of the drug. The differential effect of cycloheximide inhibition of protein synthesis on in vivo and in vitro rRNA synthesis suggests the existence of elongation control protein(s) characterized by a rapid turnover and a loose association with the nucleus.

Transcription of ribosomal DNA intergenic spacer sequences in normal and regenerating rat liver.

In regenerating rat liver both the transcriptional activity of the intergenic spacer rDNA promoter and the steady-state abundance of spacer transcripts are increased about 2-fold, as compared to normal liver. These changes are parallel to the observed 2.5-fold increase in regenerating liver of rRNA gene promotor activity and gene promotor transcripts abundance. These results suggest that both gene and spacer rDNA promotors are subject to common regulatory mechanisms. Our results indicate also that the stability of spacer transcripts in regenerating liver is not significantly altered.

Isolation of active transcription complexes from animal cell nuclei by nitrocellulose binding.

A method for the rapid isolation of active transcription complexes from animal cell nuclei is described. The method is based on the observation that, after lysis of nuclei with the detergents Sarkosyl and Triton X-100, transcription complexes are selectively bound to nitrocellulose. The nitrocellulose filters retain 80-90% o the RNA labelled briefly in vitro and about 10% of the nuclear DNA. The bulk of the retained DNA is in the size range of 20 kb. Transcription complexes involving both RNA polymerase I and II are retained by nitrocellulose. The nitrocellulose-bound transcription complexes preserve almost all of their RNA polymerase activity. The size distribution of the RNA product shows that bound transcription complexes retain also most of their growing RNA chains. The possibility to use selective retention by nitrocellulose in the analysis of transcriptionally active genes is discussed.

Limited variability of CTG/CAG repeats in Lycopersicon nuclear DNA

Seven clones containing (CTG)n/(CAG)n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.