Dynamical analysis of gene networks requires both mRNA and protein expression information

One of the important goals of biology is to understand the relationship between DNA sequence information and nonlinear cellular responses. This relationship is central to the ability to effectively engineer cellular phenotypes, pathways, and characteristics. Expression arrays for monitoring total gene expression based on mRNA can provide quantitative insight into which gene or genes are on or off; but this information is insufficient to fully predict dynamic biological phenomena. Using nonlinear stability analysis we show that a combination of gene expression information at the message level and at the protein level is required to describe even simple models of gene networks. To help illustrate the need for such information we consider a mechanistic model for circadian rhythmicity which shows agreement with experimental observations when protein and mRNA information are included and we propose a framework for acquiring and analyzing experimental and mathematically derived information about gene networks.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Interferon-alpha (Intron A) upregulates urokinase-type plasminogen activator receptor gene expression

The regulation of urokinase plasminogen activator receptor (uPAR) gene expression by interferon-alpha (IFN-alpha, or Intron A) and interferon-gamma (IFN-gamma) was studied in a HCT116 colon cancer cell line. uPAR mRNA levels were increased in a dose- and time-dependent manner in cells stimulated with IFN-alpha or IFN-gamma. uPAR protein levels reflected IFN-alpha and IFN-gamma induction of uPAR mRNA production. Cycloheximide, a protein synthesis inhibitor, also induced uPAR mRNA accumulation either alone or in combination with IFN-alpha or IFN-gamma, suggesting that the effect on uPAR mRNA levels activated by IFN-alpha or IFN-gamma does not require de novo protein synthesis. Both sodium butyrate and amiloride inhibited the uPAR mRNA levels induced by IFN-alpha or IFN-gamma. These results may provide useful information for the treatment of patients receiving IFN-alpha or IFN-gamma.     

A real-time and dynamic biological information retrieval and analysis system (BIRAS)

The aim of this study is to design a biological information retrieval and analysis system (BIRAS) based on the Internet. Using the specific network protocol, BIRAS system could send and receive information from the Entrez search and retrieval system maintained by National Center for Biotechnology Information (NCBI) in USA. The literatures, nucleotide sequence, protein sequences, and other resources according to the user-defined term could then be retrieved and sent to the user by pop up message or by E-mail informing automatically using BIRAS system. All the information retrieving and analyzing processes are done in real-time. As a robust system for intelligently and dynamically retrieving and analyzing on the user-defined information, it is believed that BIRAS would be extensively used to retrieve specific information from large amount of biological databases in now days. The program is available on request from the corresponding author.     

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.     

Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Effects of protein deprivation on alpha1(I) and alpha1(III) collagen and its degrading system in rat skin

Protein malnutrition affects the status of dermal collagen, the major structural protein in the skin. However, the molecular mechanism underlying the alteration of collagen fibers in the skin by protein deficiency remains unknown. In the present study, the effect of dietary protein deprivation on collagen metabolism was studied by analyzing the status of the synthesis and degradation of collagen in the dorsal skin of rats. Feeding on a protein-free diet for 8 days caused a dramatic decrease in both types I and III tropocollagen with a concomitant decrease in their mRNA levels, with type III collagen being more severely affected. The active form of collagenase was significantly decreased by protein deprivation, whereas the latent form was not affected. The mRNA levels of collagenase and its inhibitors (TIMP-1 and 2) were also decreased by protein deprivation. These results suggest that both the synthesis and degradation of types I and III collagen were affected by protein deficiency.     

What affects mRNA levels in leaves of field-grown aspen? A study of developmental and environmental influences

We have analyzed the abundance of mRNAs expressed from 11 nuclear genes in leaves of a free-growing aspen (Populus tremula) tree throughout the growing season. We used multivariate statistics to determine the influence of environmental factors (i.e. the weather before sampling) and developmental responses to seasonal changes at the mRNA level for each of these genes. The gene encoding a germin-like protein was only expressed early in the season, whereas the other tested genes were expressed throughout the season and showed mRNA variations on a day-to-day basis. For six of the genes, reliable models were found that described the mRNA level as a function of weather, but the leaf age was also important for all genes except one encoding an early light-inducible protein (which appeared to be regulated purely by environmental factors under these conditions). The results confirmed the importance of several environmental factors previously shown to regulate the genes, but we also detected a number of less obvious factors (such as the variation in weather parameters and the weather of the previous day) that correlated with the mRNA levels of individual genes. The study shows the power of multivariate statistical methods in analyzing gene regulation under field conditions.     

Synonymous codon usage influences the local protein structure observed

Translation of mRNA into protein is a unidirectional information flow process. Analysing the input (mRNA) and output (protein) of translation, we find that local protein structure information is encoded in the mRNA nucleotide sequence. The Coding Sequence and Structure (CSandS) database developed in this work provides a detailed mapping between over 4000 solved protein structures and their mRNA. CSandS facilitates a comprehensive analysis of codon usage over many organisms. In assigning translation speed, we find that relative codon usage is less informative than tRNA concentration. For all speed measures, no evidence was found that domain boundaries are enriched with slow codons. In fact, genes seemingly avoid slow codons around structurally defined domain boundaries. Translation speed, however, does decrease at the transition into secondary structure. Codons are identified that have structural preferences significantly different from the amino acid they encode. However, each organism has its own set of ‘significant codons’. Our results support the premise that codons encode more information than merely amino acids and give insight into the role of translation in protein folding.     

The 5'' untranslated region of mRNA for ribosomal protein S19 is involved in its translational regulation during Xenopus development.

During Xenopus development, the synthesis of ribosomal proteins is regulated at the translational level. To identify the region of the ribosomal protein mRNAs responsible for their typical translational behavior, we constructed a fused gene in which the upstream sequences (promoter) and the 5' untranslated sequence (first exon) of the gene coding for Xenopus ribosomal protein S19 were joined to the coding portion of the procaryotic chloramphenicol acetyltransferase (CAT) gene deleted of its own 5' untranslated region. This fused gene was introduced in vivo by microinjection into Xenopus fertilized eggs, and its activity was monitored during embryogenesis. By analyzing the pattern of appearance of CAT activity and the distribution of the S19-CAT mRNA between polysomes and messenger ribonucleoproteins, it was concluded that the 35-nucleotide-long 5' untranslated region of the S19 mRNA is able to confer to the fused S19-CAT mRNA the translational behavior typical of ribosomal proteins during Xenopus embryo development.     

未折叠蛋白反应—细胞内信号传导新途径

真核细胞中,当未折叠的蛋白在内质网上增多的时候,一系列内质网居民蛋白基因的转录也随之增加,这称为未折叠蛋白反应（unfolded protein response,UPR)。在酵母细胞中未折叠蛋白的感受器是Irelp蛋白,它能检测到未折叠蛋白的聚集,并将信号传递到细胞核内,诱导UPR特异转录因子Haclp mRNA的剪接成熟。成熟的Haclp蛋白能通过与UPR元件（UPR－element)的结合诱导含有这一元件的基因转录,从而启动UPR。在UPR信号传递途径中,磷酸化的Irelp与Gcn5/Ada复合物可通过解开染色体促进Haclp活性的发挥,而Ptc2p能通过使Irelp去磷酸化而反向调节UPR。目前发现UPR与磷脂生物合成存在交叉的共同途径,人类中也存在Irelp的类似物。