Gene expression profiling of B lymphocytes and plasma cells from Waldenström's macroglobulinemia: comparison with expression patterns of the same cell counterparts from chronic lymphocytic leukemia, multiple myeloma and normal individuals.

The tumoral clone of Waldenström's macroglobulinemia (WM) shows a wide morphological heterogeneity, which ranges from B lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell counterparts from chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes downregulated in WM-BL were IL4R, which plays a relevant role in CLL B-cell survival, and BACH2, which participates in the development of class-switched PC. Interestingly, one of the upregulated genes in WM-BL was IL6. A set of four genes was able to discriminate clonal BL from WM and CLL: LEF1 (WNT/beta-catenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5, which was overexpressed in WM-PC, and IRF4 and BLIMP1, which were underexpressed. In addition, three of the target genes activated by PAX5 - CD79, BLNK and SYK - were upregulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell counterpart.

Gene expression profile reveals deregulation of genes with relevant functions in the different subclasses of acute myeloid leukemia.

Bone marrow samples from 43 adult patients with de novo diagnosed acute myeloid leukemia (AML)--10 acute promyelocytic leukemias (APL) with t(15;17), four AML with inv(16), seven monocytic leukemias and 22 nonmonocytic leukemias--were analyzed using high-density oligonucleotide microarrays. Hierarchical clustering analysis segregated APL, AML with inv(16), monocytic leukemias and the remaining AML into separate groups. A set of only 21 genes was able to assign AML to one of these three classes: APL, inv(16) and other AML subtype without a specific translocation. Quantitative RT-PCR performed for 18 out of these predictor genes confirmed microarray results. APL expressed high levels of FGF13 and FGFR1 as well as two potent angiogenic factors, HGF and VEGF. AML with inv(16) showed an upregulation of MYH11 and a downregulation of a gene encoding a core-binding factor protein, RUNX3. Genes involved in cell adhesion represented the most altered functional category in monocytic leukemias. Two major groups emerged from the remaining 22 AML: cluster A with 10 samples and cluster B with 12. All the eight leukemias that were either refractory to treatment or that relapsed afterwards were assigned to cluster B. In the latter cluster, CD34 upregulation and serine proteases downregulation is consistent with a maturation arrest and lack of granulocytic differentiation.

Deregulation of microRNA expression in the different genetic subtypes of multiple myeloma and correlation with gene expression profiling.

Specific microRNA (miRNA) signatures have been associated with different cytogenetic subtypes in acute leukemias. This finding prompted us to investigate potential associations between genetic abnormalities in multiple myeloma (MM) and singular miRNA expression profiles. Moreover, global gene expression profiling was also analyzed to find correlated miRNA gene expression and select miRNA target genes that show such correlation. For this purpose, we analyzed the expression level of 365 miRNAs and the gene expression profiling in 60 newly diagnosed MM patients, selected to represent the most relevant recurrent genetic abnormalities. Supervised analysis showed significantly deregulated miRNAs in the different cytogenetic subtypes as compared with normal PC. It is interesting to note that miR-1 and miR-133a clustered on the same chromosomal loci, were specifically overexpressed in the cases with t(14;16). The analysis of the relationship between miRNA expression and their respective target genes showed a conserved inverse correlation between several miRNAs deregulated in MM cells and CCND2 expression level. These results illustrate, for the first time, that miRNA expression pattern in MM is associated with genetic abnormalities, and that the correlation of the expression profile of miRNA and their putative mRNA targets is useful to find statistically significant protein-coding genes in MM pathogenesis associated with changes in specific miRNAs.

Heterologous protein secretion directed by a repressible acid phosphatase system of Kluyveromyces lactis: characterization of upstream region-activating sequences in the KIPHO5 gene.

Transcription of the repressible acid phosphatase gene (KIPHO5) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPHO5 expression and to design new expression-secretion systems in K. lactis. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions -430 (USA1) and -192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPHO5 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (tGH-II), the promoter and signal peptide-encoding region of the phosphate-repressible KIPHO5 gene drives the expression of this gene and the secretion of the tGHII protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the Pi present in the medium; depression of heterologous protein expression can therefore be achieved by lowering the Pi concentration.

Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants.

We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088c delta and ydl084w delta haploid strains are viable in the CEN. PK2 genetic background although ydl084w delta grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects.

Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis.

The DAO1 gene of Trigonopsis variabilis encoding a D-amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D-amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N-terminal section--the FAD binding site--and the other near the C-terminal region that contains conserved signatures found in all the D-amino acid oxidases. The three C-terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D-methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild-type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D-amino acid oxidases at industrial level.

Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.