High throughput analysis of 10 microsatellite and 11 diallelic polymorphisms on the human Y-chromosome.

We describe an integrated approach to the determination of complex Y chromosome haplotypes that is both fast and relatively inexpensive. The method employs GeneScan technology to enable a researcher to assay repeat number variation at ten microsatellite loci and determine the status of 11 diallelic polymorphisms. The method requires only four PCRs and four GeneScan runs per sample and is relatively insensitive to sample DNA concentration.

Requirement for Rho in integrin signalling.

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.

Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin.

The small GTP-binding protein Rho rapidly stimulates the formation of focal adhesions and actin stress fibres when microinjected into serum-starved Swiss 3T3 fibroblasts. This response is inhibited by tyrosine kinase inhibitors. Addition of growth factors such as lysophosphatidic acid and bombesin to Swiss 3T3 cells stimulates a similar response, which is dependent on endogenous Rho proteins. To investigate signalling events regulated by Rho, we have scrape loaded Rho into serum-starved cells. Activated Rho stimulates the tyrosine phosphorylation of a number of proteins, including three proteins known to localise to focal adhesions, pp125FAK, p130 and paxillin. Rho-induced phosphorylation of pp125FAK, p130 and paxillin is observed in the absence of stress fibre formation and is, therefore, independent of Rho-induced actin polymerisation. We propose that the tyrosine kinase, pp125FAK, and the putative adapter proteins, paxillin and p130, are components of a Rho-regulated signal transduction pathway, and that these protein tyrosine phosphorylation events are likely to be important for the regulation of focal adhesion formation.

Expression of a hydrophilic surface protein in infective stages of Leishmania major.

A family of differentially expressed genes from Leishmania major contains one sequence (Gene B) that encodes a novel, hydrophilic protein found on the surface of infective parasite stages. The 177-residue, acidic Gene B protein is characterised by an amino acid repetitive element, comprising 45% of the total molecule, that is related to the cell-wall binding domain of protein A from Staphylococcus aureus. No identifiable signal peptide, membrane-spanning domain or consensus for glycosylphosphatidylinositol anchor attachment to the cell surface is found elsewhere in the deduced protein sequence. In vitro, the Gene B protein fractionates with the parasite cell surface glycoconjugates, lipophosphoglycan and the glycoinositolphospholipids. This protein is the first characterised surface peptide marker for infective stages of the Leishmania life cycle.

Genomic organisation and expression of a differentially-regulated gene family from Leishmania major.

We have isolated and characterised a differentially-regulated gene family in the protozoan parasite Leishmania major. The family contains 5 genes linked within a 10Kb region of the genome: three of the genes are closely related in DNA sequence, the other two have only limited homology. Post-transcriptional control of the differential expression pattern is suggested by detection of precursor RNA molecules containing intergenic sequences and evidence that mature mRNA molecules contain a 35nt spliced leader sequence at their 5' ends. These features support a model of polycistronic transcription in which the stability and differential processing of precursor RNA molecules determine the steady state levels of mature mRNA. We have identified several DNA sequence motifs within the gene family that have potential roles in differential processing and/or RNA stability: an alternative 5' splice acceptor site for trans-splicing; a putative polyadenylation site; and a region of potential secondary structure within 3' flanking sequences. The 3' sequence elements are conserved in those genes that share the same pattern of differential regulation. To our knowledge, this is the first example of coordinated differential-regulation of a non-identical gene cluster in Leishmania.