CAI Value Research Articles

Synthesis, cloning and expression of a novel pre-miniproinsulin analogue gene in Escherichia coli

In the present study, a novel pre-miniproinsulin analogue was designed to have a short 9 residue sequence replacing the 35 residue C-chain, one lysine and one arginine added to the C-terminus of the B-chain in combination with glycine and arginine substitution at A21 and B29, respectively, and a 16-residue fusion partner comprising the pentapeptide sequence (PSDKP) of the N-terminus of human tumor necrosis factor-α (TNF-α), 6 histidine residues for Ni2+ chelated affinity purification and a pentapeptide ending with methionine for ease of chemical cleavage fused at the N-terminus. Homology modeling of the designed protein against miniproinsulin (protein databank file 1 efeA) as a template showed that the distance between the α-carbons of the C-terminus of the B-chain and the N-terminus of the A-chain did not change; the root-mean-square deviation of the backbone atoms between the structures of modeled miniproinsulin and miniproinsulin template was 0.000Å. DNA sequencing of the synthesized gene showed 100% identity with theoretical sequence. The gene was constructed taking into account the codon preference of Escherichia coli (CAI value 0.99) in order to increase the expression rate of the DNA in the host strain. The designed gene was synthesized using DNA synthesis technology and then cloned into the expression plasmid pET-24a(+) and propagated in E. coli strain JM109. Gene expression was successful in two E. coli strains: namely JM109(DE3) and BL21(DE3)pLysS. SDS–PAGE analysis was carried out to check protein size and to check and optimize expression. Rapid screening and purification of the resulting protein was carried out by Ni–NTA technology. The identity of the expressed protein was verified by immunological detection method of western blot using polyclonal rabbit antibody against insulin.

Analysis of Codon Usage in Herpesvirus Glycoprotein B (gB) Gene

Due to the degeneracy of genetic code, most amino acids are coded by more than one codon (synonymous codons). The synonymous codons are not used at equal frequencies both within and between organisms. Of the total 33 herpesvirus gB genes, approximately 9.1% of the total gB genes had low codon bias (ENC<35), 72.7% of the gB genes had high ENC values (ENC>50), indicating that these gB genes had random codon usage in herpesviruses. There might be no direct correlation between the codon usage bias and the host, which indicates that the tRNA abundance of the host was not the main factor influencing the codon usage bias. A plot of ENC vs. GC3 indicates that mutational bias may be a more important factor than tRNA abundance in determining codon usage bias of herpesvirus gB genes. Pearson correlation coefficients between the ENC value and corresponding GC%, cumulative GC% in 2nd (GC2%) and 3rd codon position (GC3%) of each herpesvirus gB gene were -0.621 (p<0.01), -0.656 (p<0.01) and -0.712 (p<0.01), respectively, which implies that significant correlations existed between them. But no significant correlations existed between ENC and cumulative GC% in 1st codon position of each herpesviral gB gene. Furthermore, significant correlations also existed between GC% and GC3% of 33 herpesvirus gB genes (r=0.856, p<0.01). So it seems that, GC content, and particularly GC content at the 3rd base position, contributing greatly to the effective number of codons, indicating that the mutational bias dominates over translational selection. Further analysis on the relationship between gene length and ENC of 33 herpesvirus gB genes demonstrated that the two factors were not correlated. Significant correlations were found between the gene expression levels assessed by CAI value and ENC (r = -0.424, p<0.05) and GC3 values (r = 0.644, p<0.01).

Two highly homologous putative DNA-binding proteins in yeast and E. coli

WrbA [tryptophan oar) repressor-binding protein A] is a new 21 kDa protein found in some preparations of Escherichia coil iryp~ophan repressor (TrpR) l which can stimulate binding of TrpR to DNA. By searching the Swissprot database with the program FASTA 2, we found that WrbA has a highly homologous counterpart, YCP4, in the yeast Saccharomyces cerevisiae. YCP4 was identified as the hypothetical protein encoded by open reading frame (ORF) YCR042, mapping in the CDCIO--CIT2 intergenic region of yeast chromosome III (Ref. 3). WrbA and YCP4 contain 198 and 247 amino acids, respectively, as deduced from their nucleotide sequences. The two proteins show 46% identity and 65% similarity over 197 amino acids aligned with six residues gapped (Fig. I). These values strongly suggest a structural homology between the two proteins. No biochemical information regarding YCP4 is available to demonstrate that the protein is actually expressed. Neverthe- less, analysis of the promoter region and the calculation of a codon adaptation index (CAD equal to 0.2 had previously suggested that ORF YCR042 is transcribed and translated in vivo 3. This hypothesis is now strengthened by the observation of a strong evolutionary conservation, By uslng the Sharp and Li method 4, we calculated a CAI value for WrbA equal to 0.43, suggesting that this protein is also expressed with high efficiency, The calculated pl for WrbA is 5.7, while for the aligned region of YCP4 (residues 1-202) it is 6.5. For full-length YCP4 the calculated pl is 8 ~, rcficcting a carbo~-terminal extension rich in basic residues (Fig. I). A noteworthy feature of the aligned WrbA and YCP4 ~equences is the presence of a helix-turn-helix (HTH) DNA- binding motif in the carboxy-terminal region (Fig. I). WrbA fits, without mismatches, the motif AX3GXs(I/L/V ) defined by the frequency matrix of Dodd and Egan 5, while YCP4 shows a threonine re::idue in the last position of the motif. Although this substitution is not found among the known sequences of the HTH family, alanine can be observed in this position 5. Secondary structure prediction according to two different methods ~.7 detects a strong helical propensity in the regions adjacent to the central glycine of the motif in both sequences (WrbA, 177-183 and ]89--195; YCP4, 182-189 a;rd 195--207) (Fig. I). These features of WrbA and YCP4 sequences make them possible new members of the HTH family of DNA-

Infrared spectroscopy of Triassic conodonts: a new tool for assessing conodont diagenesis

The Fourier transform infrared spectra of individual conodonts with various CAI values indicate definite changes in conodont francolite during diagenesis. Steady decarbonation can be observed in the increasing intensity of the band with the wavenumber 2340 an-1, which is assigned to trapped molecular CO2. Carbon dioxide originates from decomposing CO2-3-ions occupying the B-site in francolite. Furthermore, the intensity of the water-deformation band at 1644 cm-1 decreases with higher CAI values. These changes of intensity can be quantified by calculating the maximum extinction (Emax) for the corresponding absorption maxima and correlated with the CAI. Unaltered conodonts have a relative variable water ‘content’ and very little CO2. Up to a CAI value of 4 conodont francolite continuously expells water and carbon dioxide is trapped. Conodonts with CAI = 5 have similar water ‘contents’ to CAI = 4 conodonts, but decarbonation continues to take place during this stage of diagenesis.

Kinetics of Cytosolic Calcium and Aldosterone Responses in Rat Adrenal Glomerulosa Cells*

To evaluate the relationship between cytosolic calcium (Cai) and aldosterone production, rat adrenal zona glomerulosa (ZG) cells were studied during long-term stimulation by different secretagogues. Cai was measured in single ZG cells using microspectrofluorimetry, and aldosterone was determined in cell populations using a superfusion system. For external potassium (K+), Cai increases are sustained, with only a slight decrement over time, a feature shared by aldosterone production. The relationship between aldosterone output and Cai is nonlinear, with a Cai value for half-maximal stimulation of approximately 500 nM. Furthermore, the sustained changes in Cai with external K+ indicate that ZG cells can use an amplitude-based Cai signal to stimulate aldosterone production. Cai changes stimulated by angiotensin-II (Ang-II) show a complex dose-response pattern, with high concentrations (greater than or equal to 1 nM) of Ang-II eliciting a peak-plateau signal and lower doses (0.1 nM to 10 pM) producing repeated Cai oscillations. The peak amplitude of the Cai response in individual cells is not dose dependent, with the ZG cell experiencing peak levels repeatedly at the lowest Ang-II concentrations. However, the Cai transients are more frequent with increasing Ang-II concentrations between 0.1 nM and 10 pM. When integrated over time, the mean Cai signal also shows only modest dose-dependency during the sustained phase of Ang-II stimulation. Unlike the integrated Cai signal, aldosterone production increases steeply between 10 pM and 0.1 nM Ang-II, indicating that the Cai signal is likely to be frequency-based. Conversely, the steroid response to high Ang-II closely mirrors the kinetics of the more sustained Cai signals, including the diminished Cai and aldosterone levels during sustained stimulation with the highest Ang-II doses. Arginine vasopressin stimulated Cai and aldosterone responses, which closely resemble those elicited by 0.1 nM Ang-II, except that both Cai and aldosterone return to basal values within 20 min of continuous presentation of arginine vasopressin. Each ZG secretagogue produces a distinct pattern of Cai and aldosterone response. In addition, Cai response patterns can be divided into two general classes: a sustained Cai response, which appears to modulate cell activation by the amplitude of the Cai signal, and an oscillating Cai response, which uses the frequency of the Cai transients to control the magnitude of stimulation.