Chriz, a chromodomain protein specific for the interbands of Drosophila melanogaster polytene chromosomes.

Polytene interphase chromosomes are compacted into a series of bands and interbands reflecting their organization into independent chromosomal domains. In order to understand chromosomal organization, we set out to study the role of proteins that are selective for interbands. Here we describe the Drosophila melanogaster chromodomain protein Chriz that is coimmunoprecipitated with the zinc finger protein Z4. Both proteins colocalize exclusively to the interbands on Drosophila polytene chromosomes. Like Z4, Chriz is ubiquitously expressed throughout development and is associated with chromatin in all interphase nuclei. Following dissociation from chromatin, early in mitosis Chriz binds to the centrosomes and to the mitotic spindle. Newly induced amorphic Chriz alleles are early lethal, and ubiquitous overexpression of Chriz is lethal as well. Available Chriz hypomorphs which survive until pupal stage have a normal chromosomal phenotype. Reducing Z4 protein does not affect Chriz binding to polytene chromosomes and vice versa. Z4 is still chromosomally bound when Chriz protein is depleted by RNA interference.

Identification of acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

Matrix-assisted laser desorption/ionization-mass spectrometry peptide mass mapping and nano-electrospray ionization tandem mass spectrometry were used to identify acidic, low molecular mass proteins of Mycobacterium tuberculosis strain H37Rv. Proteins were extracted from whole cell lysates of mycobacteria, separated by high resolution two-dimensional electrophoresis (2-DE) and analysed by mass spectrometry (MS). Silver-stained 2-DE patterns resolved about 1800 distinct protein species, 190 of which had an observed isoelectric point and molecular mass in the range of pH 4 to 6 and 6 to 15 kDa, respectively. Seventy-six spots from this range were excised from Coomassie Brilliant Blue G250-stained gels and analysed by MS, from which 72 were identified. These spots were shown to represent products of as many as 50 different protein-coding genes. Ten genes gave rise to more than one protein species. Eleven spots contained more than one protein. The present study led to the identification of 15 mycobacterial proteins with assigned putative functions, 28 conserved hypothetical proteins and one unknown protein. Most proteins of the latter two groups had previously been predicted at the DNA level only. Six additional spots were shown to comprise proteins encoded by open reading frames that have not been predicted for M. tuberculosis H37Rv by genomic investigations.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Proteomics reveals open reading frames in Mycobacterium tuberculosis H37Rv not predicted by genomics.

Genomics revealed the sequence of 3924 genes of the H37Rv strain of Mycobacterium tuberculosis. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.

Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.

The RRM protein NonA from Drosophila forms a complex with the RRM proteins Hrb87F and S5 and the Zn finger protein PEP on hnRNA.

The RRM protein NonA, an ubiquitous nuclear protein present in puffs on polytene chromosomes, has been immunopurified as a RNA-protein complex from Drosophila Kc cells. Three other proteins present in the complex have been identified: X4/PEP (protein on ecdysone puffs), a 100-kDa zinc finger RNA-binding protein; the 70-kDa S5 protein, an as yet uncharacterized RNA-binding protein; and P11/Hrb87F, a 38-kDa RRM protein homologous to hnRNP protein A1 from mammals. Monoclonal antibodies against any of the protein components coprecipitate all four proteins although at different ratios. NonA does not coprecipitate with the hrp40 hnRNP proteins and immunolocalizes in a pattern distinct of major hnRNP proteins. Like NonA, X4/PEP, S5, and P11/Hrb87F are present on active sites on polytene chromosomes. The precipitated NonA complex is enriched for certain protein encoding RNAs, notably, histone H3 and H4 RNA.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.