Characterization and hepatic expression of rat alpha 1-inhibitor III mRNA

A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA library constructed in the expression vector lambda gt11. M13 vector sequence analysis yielded a deduced protein primary structure that was 89% homologous to the prototype alpha 1-inhibitor III (alpha 1I3) sequence presented in the preceding paper by Braciak et al. (Braciak, T. A., Northemann, W., Hudson, G. O., Shields, B. R., Gehring, M. R., and Fey, G. H. (1988) J. Biol. Chem. 263, 3999-4012) with regard to exact matches and 92% homologous when considering chemically conserved residues. The clone also possessed 100% homology to the putative bait region of a variant clone (pRLA1I3/27J) of alpha 1I3. Such sequence data demonstrates that the AF7 clone corresponds to a member of the family of variant alpha 1I3 mRNAs. Furthermore, this report presents the entire mRNA sequence corresponding to the 3'-half of alpha 1I3 variant 27J. We have utilized AF7 cDNA to study the expression of alpha 1I3 messenger RNA encoding this liver-specific glycoprotein under conditions known to alter hepatic gene expression. Our data reveal that alpha 1I3 mRNA expression is not only regulated during the acute-phase response but is also modulated in response to a variety of changing physiological conditions, most notably liver development. Steady state levels of mRNA were quantified using Northern blot techniques and laser densitometry. During acute phase response initiated by turpentine injection, the relative abundance of alpha 1I3 mRNA decreased 4-5-fold over a period of 24 h. Following partial hepatectomy, the regenerating liver expressed six-fold less alpha 1I3 mRNA than untreated liver after 24 h. This reduced level was maintained over a 2-day period. We have also demonstrated that alpha 1I3 mRNA expression is developmentally regulated. Fetal rat liver did not contain detectable concentrations of rat alpha 1I3 mRNA even as late as 4 days prior to birth. However, trace amounts were observed from birth until approximately 20 days of age when alpha 1I3 mRNA levels increased 10-fold to maximal adult quantities over the following 2 or 3 weeks. During the course of pregnancy, alpha 1I3 mRNA remained essentially constant until approximately 4 days prior to birth when a precipitous decline to 40% of the original level was noted. Subsequently, normal values were gradually restored over a 30-day postpartum period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a glucocorticoid response element in the 3'-flanking region of the human Dexras1 gene

The Dexras1 gene responds to glucocorticoids with a rapid and profound induction. A glucocorticoid response element (GRE) was identified in the 3'-flanking region (2.3 kb downstream of poly(A) signal) of the human Dexras1 gene. This element conferred rapid glucocorticoid responsiveness when inserted into a homologous promoter-driven luciferase reporter. A point mutation within the 15-bp GRE abolished this glucocorticoid responsiveness.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

大鼠肝癌中α—抑制因子3基因表达及基因结构的...

The expression and structure of alpha 1-inhibitor 3 (alpha 1-I3) gene were investigated in 16 primary rat hepatomas induced by diethylnitrosamine. In most tumor samples (75%, 12/16), the expression of alpha 1-I3 gene manifested markedly diminished or undetectable level of alpha 1-I3 mRNA. Further study indicated that the abnormal expression of alpha 1-I3 gene in the hepatomas examined might be due to, at least in part, the gene hypermethylation, insertion of repeat sequence(s) or base substitutions at the recognition sequences of some restriction endonucleases which caused certain alteration in the gene structure. The significance of alpha 1-I3 as an endogenous antitumor factor in hepatocarcinogenesis was also discussed.     

Identification of factors contributing to variability in a blood-based gene expression test

Background

Corus CAD is a clinically validated test based on age, sex, and expression levels of 23 genes in whole blood that provides a score (1–40 points) proportional to the likelihood of obstructive coronary disease. Clinical laboratory process variability was examined using whole blood controls across a 24 month period: Intra-batch variability was assessed using sample replicates; inter-batch variability examined as a function of laboratory personnel, equipment, and reagent lots.

Methods/Results

To assess intra-batch variability, five batches of 132 whole blood controls were processed; inter-batch variability was estimated using 895 whole blood control samples. ANOVA was used to examine inter-batch variability at 4 process steps: RNA extraction, cDNA synthesis, cDNA addition to assay plates, and qRT-PCR. Operator, machine, and reagent lots were assessed as variables for all stages if possible, for a total of 11 variables. Intra- and inter-batch variations were estimated to be 0.092 and 0.059 Cp units respectively (SD); total laboratory variation was estimated to be 0.11 Cp units (SD). In a regression model including all 11 laboratory variables, assay plate lot and cDNA kit lot contributed the most to variability (p = 0.045; 0.009 respectively). Overall, reagent lots for RNA extraction, cDNA synthesis, and qRT-PCR contributed the most to inter-batch variance (52.3%), followed by operators and machines (18.9% and 9.2% respectively), leaving 19.6% of the variance unexplained.

Conclusion

Intra-batch variability inherent to the PCR process contributed the most to the overall variability in the study while reagent lot showed the largest contribution to inter-batch variability.     

Estrogen receptor alpha interaction with estrogen response element half-sites from the rat prolactin gene

Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene.