Transcription-associated protein families are primarily taxon-specific.

The mechanisms controlling gene regulation appear to be fundamentally different in eukaryotes and prokaryotes (Struhl (1999) CELL, 98, 1-4). To investigate this diversity further, we have analysed the distribution of all known transcription-associated proteins (TAPs), as reflected by sequence database annotations. Our results for the primary phylogenetic domains (Archaea, Bacteria and Eukaryota) show that TAP families are mostly taxon-specific and very few transcriptional regulators are common across these domains.

Using 3' untranslated sequences to identify differentially expressed genes in Leishmania.

A more sensitive screen for Leishmania major genes differentially expressed as the insect stage develops into an infectious form (metacyclogenesis) has been devised. The screen expolits the observation that in kinetoplastid protozoa differentially expressed genes are often associated with unique 3' untranslated regions (UTRs). To obtain probes encoding this region, cDNA is synthesised using an oligo-dT primer containing the universal vectorette sequence in the first strand reaction and an oligonucleotide comprising the spliced leader sequence in the second strand reaction. The cDNAs are then cleaved with Sau3AI, ligated to the vectorette and the 3' UTRs polymerase chain reaction (PCR) amplified using the universal vectorette sequence as the primer. Differential screening with PCR-amplified 3' UTRs uncovered: (1) previously identified metacyclic-specific expressed genes; (2) cloned genes which had not been shown to be differentially regulated; and (3) a new gene identified only as a match to two identical L. major expressed sequence tags (ESTs) that is upregulated in the infectious stage.

Differential expression of Leishmania major beta-tubulin genes during the acquisition of promastigote infectivity.

Tubulin expression has been analysed as the insect stage of the protozoan parasite Leishmania major differentiates from a non-infective to an infective form. This transformation of the promastigote stage occurs in vitro and analysis of beta-tubulin mRNA expression in axenically grown promastigotes showed that a 2200 nt transcript is predominately expressed in non-infective promastigotes. The message contains a motif associated with mRNA intracellular localisation and its level is reduced by an order of magnitude in infective promastigotes through a mechanism involving RNA stability. A 3200 nt RNA, the major beta-tubulin transcript in the infective stage, is encoded by a single copy gene at the 3' end of the array that encodes the 2200 nt RNA. These RNAs, as well as a gene encoding a beta-tubulin transcript highly up-regulated in the mammalian stage of the parasite, encode polypeptides that are apparently functionally equivalent but have highly diverged 3' untranslated regions. This differential regulation of the dispersed isogenes may reflect the involvement of a mechanism altering tubulin synthesis during the Leishmania life cycle. The analysis of alpha-tubulin RNA levels revealed the abundance of this message falls as promastigotes differentiate into an infectious stage and the transcript is destabilised in infective promastigotes. These data demonstrate that the regulation of mRNA half-life contributes to controlling gene expression as promastigotes differentiate into an infectious form.

An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis.

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.

Ferritin synthesis is controlled by iron-dependent translational derepression and by changes in synthesis/transport of nuclear ferritin RNAs.

Ferritin synthesis, maintained at a very low basal rate when extracellular iron levels are low, is elevated up to 50-fold when iron levels are increased. Previous examinations of this iron-dependent activation have concluded that changes in ferritin synthesis results from selective translational activation conferred by an "iron response element" that lies near the 5' terminus of all known ferritin mRNAs. By placing an iron response element in an optimal position in other mRNAs, we find the iron response element to be a potent translational repressor whose influence can only partially be abrogated under optimal inducing conditions. Further, we show that the 25- to 50-fold iron-mediated increase in ferritin synthesis results from coupling a 5- to 6-fold iron-dependent translational derepression with a similar 5- to 6-fold nuclear-dependent increase in mRNA level.

Amplification and analysis of human DNA present in mosquito bloodmeals.

DNA fingerprinting should permit the identification of individual human hosts of haematophagous arthropods, providing epidemiologically useful information, for example, the biting rates on different people and the impact of insecticide-impregnated bednets. Investigations reported here demonstrate that it is possible to extract, amplify and fingerprint human DNA from the bloodmeals of individual female Anopheles gambiae mosquitoes kept at 24 degrees C for up to 10-15 h post-ingestion.

Isolation of genes showing increased or unique expression in the infective promastigotes of Leishmania major.

The trypanosomatid parasite Leishmania major is one of the principal causal agents of human cutaneous leishmaniasis. Promastigotes grown in vitro undergo growth cycle-dependent differentiation, associated with morphological and biochemical changes, to produce forms which are infective to the mammalian host. By differentially screening a cDNA library constructed from stage-specific mRNA, we have isolated 4 clones encoding mRNAs which show unique or elevated expression in the infective promastigotes of Leishmania major. One of these clones is homologous to a heat-shock protein 70-related gene, that is non-heat-inducible but shows up-regulation during promastigote differentiation. Each of the other cDNAs isolated also recognises multiple transcripts, which show differential regulation between parasite stages and are encoded by repeated, linked nuclear genes. In trypanosomatids, this genomic organisation is indicative of polycistronic transcription.

A family of heat shock protein 70-related genes are expressed in the promastigotes of Leishmania major.

We describe the isolation and characterisation of two novel genes of the parasitic protozoan Leishmania major that are related by nucleotide sequence homology to eukaryotic genes encoding 70 Kd. heat shock proteins. The transcription of neither gene is heat-inducible but both are constituitively-expressed throughout the promastigote stage of the parasite life cycle. A third gene shows differential expression between non-infective and infective promastigote stages in the absence of any temperature change. These genes are related by sequence homology to the tandemly-repeated hsp70 genes of trypanosomatids, but are located on different, dispersed chromosomes within the genome of L. major. The open reading frame for translation derived from one of these sequences contains a putative mitochondrial signal peptide at its amino-terminus.