Identification of proteins whose synthesis is modulated during the cell cycle of Saccharomyces cerevisiae

We examined the synthesis and turnover of individual proteins in the Saccharomyces cerevisiae cell cycle. Proteins were pulse-labeled with radioactive isotope (35S or 14C) in cells at discrete cycle stages and then resolved on two-dimensional gels and analyzed by a semiautomatic procedure for quantitating gel electropherogram-autoradiographs. The cells were obtained by one of three methods: (i) isolation of synchronous subpopulations of growing cells by zonal centrifugation.; (ii) fractionation of pulse-labeled steady-state cultures according to cell age; and (iii) synchronization of cells with the mating pheromone, alpha-factor. In confirmation of previous studies, we found that the histones H4, H2A, and H2B were synthesized almost exclusively in the late G1 and early S phases. In addition, we identified eight proteins whose rates of synthesis were modulated in the cell cycle, and nine proteins (of which five, which may well be related, were unstable, with half-lives of 10 to 15 min) that might be regulated in the cell cycle by periodic synthesis, modification, or degradation. Based on the time of maximal labeling in the cell cycle and on experiments with alpha-factor and hydroxyurea, we assigned the cell cycle proteins to two classes: proteins in class I were labeled principally in early G1 phase and at a late stage of the cycle, whereas those in class II were primarily synthesized at times ranging from late G1 to mid S phase. At least one major control point for the cell cycle proteins occurred between "start" and early S phase. A set of stress-responsive proteins was also identified and analyzed. The rates of synthesis of these proteins were affected by certain perturbations that resulted during selection of synchronous cell populations and by heat shock.     

Identification of essential alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

The cell cycle of Caulobacter crescentus is controlled by a complex signalling network that co‐ordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non‐essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.     

Identification of a core set of genes that signifies pathways underlying cardiac hypertrophy

Although the molecular signals underlying cardiac hypertrophy have been the subject of intense investigation, the extent of common and distinct gene regulation between different forms of cardiac hypertrophy remains unclear. We hypothesized that a general and comparative analysis of hypertrophic gene expression, using microarray technology in multiple models of cardiac hypertrophy, including aortic banding, myocardial infarction, an arteriovenous shunt and pharmacologically induced hypertrophy, would uncover networks of conserved hypertrophy-specific genes and identify novel genes involved in hypertrophic signalling. From gene expression analyses (8740 probe sets, n = 46) of rat ventricular RNA, we identified a core set of 139 genes with consistent differential expression in all hypertrophy models as compared to their controls, including 78 genes not previously associated with hypertrophy and 61 genes whose altered expression had previously been reported. We identified a single common gene program underlying hypertrophic remodelling, regardless of how the hypertrophy was induced. These genes constitute the molecular basis for the existence of one main form of cardiac hypertrophy and may be useful for prediction of a common therapeutic approach. Supplementary material for this article can be found at: http://www.interscience.wiley.com/jpages/1531-6912/suppmat.     

Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis.

The levels of urease and asparaginase were elevated 25- and 20-fold, respectively, in extracts of Bacillus subtilis cells grown in medium containing nitrogen sources that are poor sources of ammonium (NH4+) compared with the levels seen in extracts of cells grown in medium containing nitrogen sources that are good sources of NH4+. To determine whether a collection of genes whose expression responds to nitrogen availability could be isolated, a library of Tn917-lacZ insertions was screened for nitrogen-regulated beta-galactosidase expression. Two fusion strains were identified. beta-Galactosidase expression was 26- and 4,000-fold higher, respectively, in the nrg-21::Tn917-lacZ and the nrg-29::Tn917-lacZ insertion strains during NH4(+)-restricted growth than during growth on nitrogen sources that are good sources of NH4+. PBS1 transduction analysis showed that the nrg-21::Tn917-lacZ insertion mapped between gutB and purB and that the nrg-29::Tn917-lacZ insertion mapped between degSU and spoIID. The repression of expression of these four gene products during growth on good sources of NH4+ required the wild-type glutamine synthetase protein but not the glutamine synthetase regulatory protein, GlnR.     

Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes

Water borne cercaria(ae) of the trematode genus Schistosoma rapidly penetrate host skin. A single serine protease activity, cercarial elastase, is deposited in advance of the invading parasite by holocytosis of vesicles from ten large acetabular gland cells. Cercarial elastase activity is a composite of multiple isoforms. Genes coding for the isoforms can be divided into two classes by amino acid and promoter sequence homology. Two of the five genes identified in Schistosoma mansoni account for over 90% of the activity and protein released. The remaining genes produce little protein or are silent. Positional scanning synthetic combinatorial substrate libraries demonstrate that the two major isoforms have similar substrate specificities and are, therefore, isoenzymes. The closely related Schistosoma hematobium and the distantly related Schistosomatium douthitti also contain multiple orthologous cercarial elastase genes suggesting that gene duplication may have occurred after speciation in Schistosoma evolution and that this duplication has been conserved.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation

A 36K MW polypeptide was previously identified by two-dimensional gel electrophoresis whose relative proportion increases in S phase of HeLa cells, and following viral transformation of mouse 3T3B and hamster BHK21 cells. We report here experiments that show that this polypeptide (isoelectric focusing (IEF 49)) is localized in the nucleus and that its relative proportion decreases dramatically under conditions for which there is a decrease in the rate of cell proliferation, due for example, to the formation of giant HeLa cells by X-ray irradiation or the senescence of human skin fibroblasts. It is present in negligible amounts in non-cycling cells of adult tissues. These studies also revealed and unrelated cytoplasmic polypeptide (IEF 52; tropomyosin related; coordinates 35/1.43) whose relative proportion increased significantly and reproducibly with a decrease in the rate of cell proliferation. It is suggested that IEF 49 may be a useful marker for cycling cells.     

Identification of a set of genes with developmentally down-regulated expression in the mouse brain.

Using a subtraction cloning approach, we have isolated a set of cDNA clones from mouse neural precursor cells whose respective mRNA levels are down-regulated during the development of mouse brain. Single stranded DNA prepared from neuronal precursor cell cDNA library in lambda Zap vector was subtracted with poly (A)+ RNA prepared from postnatal and adult mouse brain to obtain several clones which show developmental down-regulation of expression. Their patterns of expression indicate that these genes may play important roles during the embryonic development and differentiation of central nervous system.     

Identification of a four immune-related genes signature based on an immunogenomic landscape analysis of clear cell renal cell carcinoma

Renal clear cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which has strong immunogenicity. A comprehensive study of the role of immune-related genes (IRGs) in ccRCC is of great significance in finding ccRCC treatment targets and improving patient prognosis. In this study, we comprehensively analyzed the expression of IRGs in ccRCC based on The Cancer Genome Atlas datasets. The mechanism of differentially expressed IRGs in ccRCC was analyzed by bioinformatics. In addition, Cox regression analysis was used to screen prognostic related IRGs from differentially expressed IRGs. We also identified a four IRGs signature consisting of four IRGs (CXCL2, SEMA3G, PDGFD, and UCN) through lasso regression and multivariate Cox regression analysis. Further analysis results showed that the four IRGs signature could effectively predict the prognosis of patients with ccRCC, and its predictive power is independent of other clinical factors. In addition, the correlation analysis of immune cell infiltration showed that this four IRGs signature could effectively reflect the level of immune cell infiltration of ccRCC. We also found that the expression of immune checkpoint genes CTLA-4, LAG3, and PD-1 in the high-risk group was higher than that in the low-risk group. Our research revealed the role of IRGs in ccRCC, and developed a four IRGs signature that could be used to evaluate the prognosis of patients with ccRCC, which will help to develop personalized treatment strategies for patients with ccRCC and improve their prognosis. In addition, these four IRGs may be effective therapeutic targets for ccRCC.