Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies.

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, peptidylprolyl isomerase A, beta 2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, beta-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD(260) measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD(260) measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.

Human disease characterization: real-time quantitative PCR analysis of gene expression.

Real-time quantitative PCR is the measurement of a fluorescent signal generated and measured during PCR as a consequence of amplicon synthesis. When used as reverse transcriptase-PCR (RT-PCR), real-time quantitative PCR has proved to be useful in accurately measuring expression levels of specific gene transcripts. When applied to questions of minimal residual disease, the technique has evolved from generically detecting the presence of disease cells in individuals, such as the AML1-ETO fusion transcript, to the identification of a specific gene, such as BCL-6, which is prognostic for determining the therapeutic outcome of patients with diffuse large B-cell lymphoma.

Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response.

NK cells constitutively express monocyte-derived cytokine (monokine) receptors and secrete cytokines and chemokines following monokine stimulation, and are therefore a critical component of the innate immune response to infection. Here we compared the effects of three monokines (IL-18, IL-15, and IL-12) on human NK cell cytokine and chemokine production. IL-18, IL-15, or IL-12 alone did not stimulate significant cytokine or chemokine production in resting NK cells. The combination of IL-18 and IL-12 induced extremely high amounts of IFN-gamma protein (225 +/- 52 ng/ml) and a 1393 +/- 643-fold increase in IFN-gamma gene expression over those in resting NK cells. IL-15 and IL-12 induced less IFN-gamma protein (24 +/- 10 ng/ml; p < 0.007) and only a 45 +/- 19-fold increase in IFN-gamma gene expression over those in resting NK cells. The CD56bright NK cell subset produced significantly more IFN-gamma following IL-18 and IL-12 compared with CD56dim NK cells (p < 0.008). However, the combination of IL-15 and IL-12 was significantly more potent than that of IL-18 and IL-12 for NK cell production of IL-10, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and TNF-alpha at the protein and transcript levels. Granulocyte-macrophage CSF was optimally induced by IL-15 and IL-18. Resting CD56+ NK cells expressed IL-18R transcript that was up-regulated by IL-12 or IL-15. Our results show that distinct cytokine and chemokine patterns are induced in NK cells in response to different costimulatory signals from these three monokines. This suggests that NK cell cytokine production may be governed in part by the monokine milieu induced during the early proinflammatory response to infection and by the subset of NK cells present at the site of inflammation.

Effects of Sin- versions of histone H4 on yeast chromatin structure and function.

Previous studies have identified single amino acid changes within either histone H3 or H4 (Sin- versions) that allow transcription in the absence of the yeast SWI-SNF complex. The histone H4 mutants are competent for nucleosome assembly in vivo, and the residues that are altered appear to define a discrete domain on the surface of the histone octamer. We have analyzed the effects of the Sin- versions of histone H4 on transcription and chromatin structure in vivo. These histone H4 mutants cause an increased accessibility of nucleosomal DNA to Dam methyltransferase and to micrococcal nuclease. Sin- derivatives of histone H4 also grossly impair the ability of nucleosomes to constrain supercoils in vivo. Nucleosome-mediated repression of the PHO5 gene is severely impaired by these histone H4 mutants; PHO5 expression is derepressed to 31% of the wild-type induced level. In contrast to the induction caused by nucleosome depletion, full PHO5 derepression by Sin- versions of histone H4 requires upstream regulatory elements. In addition, Sin- derivatives of histone H4 do not activate expression from CYC1 or GAL1 promoters that lack UAS elements. We propose that these Sin- mutations alter histone-DNA contact residues that play key roles in restricting the accessibility of nucleosomal DNA to transcription factors.

Regulation of the expression of three housekeeping genes encoding subunits of the Neurospora crassa vacuolar ATPase.

The vacuolar ATPase is a complex enzyme and is encoded by at least nine genes, which appear to be scattered throughout the genome. We have examined the vma-1 vma-2, and vma-3 genes, which encode subunits present in multiple copies within the Neurospora crassa vacuolar ATPase. We wished to see if the expression of these genes is coordinately regulated and if these genes contain similar promoter elements. A region was sequenced of approximately 1 kb located upstream of the protein coding region for each gene. Several sequence elements were found in similar positions in each of the three genes. Each of the genes had several strong transcription initiation sites, clustered within 13-60 bp and located 112-193 bp upstream of the translation start site. The size and abundance of the RNA transcripts was also determined: the amount of RNA transcribed from each gene was roughly proportional to the numbers of each subunit present in the enzyme. A series of plasmids was constructed containing parts of the putative promoter region fused to beta-galactosidase. Analysis of these plasmids indicated that the essential region of the vma promoters lies within 370 bp of the protein coding region. Overall, the vma genes appear to have similar characteristics to "housekeeping" genes described in other organisms.

The proteolipid subunit of the Neurospora crassa vacuolar ATPase: isolation of the protein and the vma-3 gene.

We have isolated the proteolipid subunit from the vacuolar ATPase of Neurospora crassa, using ion-exchange chromatography. We have also isolated several cDNA clones and the corresponding genomic DNA that encodes this subunit. The derived protein sequence indicates that the polypeptide is composed of 161 amino acid residues with an M(r) of 16,328 kDa. The gene encoding the proteolipid, named vma-3, is unusual in several respects. It contains four introns and, unlike other fungal genes, has non-coding regions that are as large as the coding regions. The 3' untranslated regions of the cDNAs were quite heterogeneous, with polyadenylation sites more than 300 bp apart. Analysis of the mRNA indicates that two size classes of transcripts are produced, differing in the length of the 3' untranslated region. Mapping of the vma-3 gene showed that it is closely linked, but not adjacent to, vma-1, the gene encoding the 67 kDa subunit of the vacuolar ATPase. This raises the possibility that in N. crassa some of the vacuolar ATPase genes may be clustered.

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1.

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.