TBP2 is a substitute for TBP in Xenopus oocyte transcription

Background  

TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.     

Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs

Background  

The canonical core promoter elements consist of the TATA box, initiator (Inr), downstream core promoter element (DPE), TFIIB recognition element (BRE) and the newly-discovered motif 10 element (MTE). The motifs for these core promoter elements are highly degenerate, which tends to lead to a high false discovery rate when attempting to detect them in promoter sequences.     

Identification of valid reference genes during the differentiation of human myoblasts

Background  

Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.     

Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1

Background  

The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells.     

Different cDNA microarray patterns of gene expression reflecting changes during metastatic progression in adenoid cystic carcinoma

Background  

The metastatic ability of tumor cells is determined by level of expression of specific genes that may be identified with the aid of cDNA microarray containing thousands of genes and can be used to establish the expression profile of disease related genes in complex biological system.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

SBEAMS-Microarray: database software supporting genomic expression analyses for systems biology

Background  

The biological information in genomic expression data can be understood, and computationally extracted, in the context of systems of interacting molecules. The automation of this information extraction requires high throughput management and analysis of genomic expression data, and integration of these data with other data types.     

Leukocyte infiltrate in gastrointestinal adenocarcinomas is strongly associated with tumor microsatellite instability but not with tumor immunogenicity

Purpose  

To analyze the correlation of genomic instability with leukocyte infiltrate in gastrointestinal carcinomas (GIACs) and with tumor immunogenicity, e.g., HLA class I cell surface expression defects and galectin-3 and PDL-1 expression.     

DISCLOSE : DISsection of CLusters Obtained by SEries of transcriptome data using functional annotations and putative transcription factor binding sites

Background  

A typical step in the analysis of gene expression data is the determination of clusters of genes that exhibit similar expression patterns. Researchers are confronted with the seemingly arbitrary choice between numerous algorithms to perform cluster analysis.     

Using mechanistic Bayesian networks to identify downstream targets of the Sonic Hedgehog pathway

Background  

The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge.     

Use of genomic DNA control features and predicted operon structure in microarray data analysis: ArrayLeaRNA – a Bayesian approach

Background  

Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge.