On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine

Beggs, J D

Brunet-Simon, A

Colley, A

Decourty, L

Dix, I

Fromont-Racine, M

Joly, N

Legrain, P

Mayes, A E

Rain, J C

Ricard, F

German

PubMed

Crossref

Current Awareness on Comparative and Functional Genomics

In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional clectrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via twodimensional gel clectrophoresis. These two methods arc the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatograpliy-tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteomc analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.7 page(s

Haynes, P A

Yates, J R

Macquarie University ResearchOnline

Yeast

Proteome profiling - Pitfalls and progress

International audienceA set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale

Fromont-Racine, Micheline

Mayes, Andrew, E.

Brunet-Simon, Adeline

Rain, Jean-Christophe

Colley, Alan

Dix, Ian

Decourty, Laurence

Joly, Nicolas

Ricard, Florence

Beggs, Jean, D.

Legrain, Pierre

HAL-Pasteur

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins

17: 95±110.associated protein with signi®cant similarity to core proteins of small nuclear ribonucleoproteins.

A comprehensive analysis of protein±protein interactions in Saccharomyces cerevisiae.

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals ®ve novel proteins.

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3k-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

A generic protein puri®cation method for protein complex characterization and proteome exploration.

A genomic approach of the hepatitis C virus generates a protein interaction map.

A protein±protein interaction map of yeast RNA polymerase III.

A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts.

A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles.

An extragenic suppressor of prp24-1 de®nes genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae.

An improved method for polymerase chain reaction using whole yeast cells.

Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding.

Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant.

Characterisation of Sm-like proteins in yeast and their association with U6 snRNA.

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Dynamic RNA-RNA interactions in the spliceosome.

eIF2 independently binds two distinct eIF2B subcomplexes that catalyze and regulate guanine-nucleotide exchange.

Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.

Functional analysis of homologs of translation initiation factor 2c in yeast.

Human snRNP polypeptide D1 promotes pre-mRNA binding in yeast and de®nes non-essential yeast Smd1p sequences.

Identi®cation and characterization of Uss1p (Sdb23p): a novel U6 snRNA108 M. Fromont-Racine et al.

Identi®cation and initial characterization of the cytosolic protein Ycr77p.

Identi®cation by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

Identi®cation of a novel component of the nonsense-mediated mRNA decay pathway by use of an interacting protein screen.

Interactions within the yeast Sm core complex: from proteins to amino acids.

Mammalian Ras interacts directly with the serine/threonine kinase Raf.

Molecular Cloning: A Laboratory Manual, 2nd edn.

Multiple protein: protein interactions between the snRNP common core proteins.

Mutations in trans-acting factors affecting mRNA decapping in Saccharomyces cerevisiae.

Mutations in translation initiation factors lead to increased rates of deadenylation and decapping of mRNAs in Saccharomyces cerevisiae.

New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.

Nucleo-cytoplasmic transport of U snRNPs: de®nition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap.

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Protein interaction mapping in Caenorhabditis elegans using proteins involved in vulval development.

PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle.

PRP4: a protein of the yeast U4/ U6 small nuclear ribonucleoprotein particle.

Puri®cation of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identi®cation of its proteins.

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

Role of the essential yeast protein PSU1 in the transcriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors.

Sm and Sm-like proteins assemble in two Genome-wide protein interaction maps in mRNA metabolism pathways 109 Copyright #

Sm and Sm-like proteins belong to a large family: identi®cation of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein±protein interactions.

snRNP structure and function,

Speci®city of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs.

Splicing of Precursors to mRNA by the Spliceosomes.

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Structure of spliceosomal snRNPs and their role in pre-mRNA splicing.

Suppressors of a U4 snRNA mutation de®ne a novel U6 snRNP protein with RNA-binding motifs.

The 3k to 5k degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3k to 5k exonucleases of the exosome complex.

The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP.

The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.

The yeast exosome and human PM-Scl are related complexes of 3k -->5k exonucleases.

The yeast SME1 gene encodes the homologue of the human E core protein.

Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens.

Toward a protein±protein interaction map of the budding yeast: a comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins.

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

U2 small nuclear RNA 3k end formation is directed by a critical internal structure distinct from the processing site.

U6 snRNA maturation and stability.

Yeast Sm-like proteins function in MRNA decapping and decay.

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