The human basement membrane specific collagen type IV is a heterotrimer composed of two alpha 1(IV) chains and one alpha 2(IV) chain. A partial genomic EcoRI library was screened with cDNA clones representing the 5′ end regions of the alpha 1(IV) and the alpha 2(IV) mRNA. A 2.2‐kb genomic fragment was isolated and sequenced, which contains the 5′ terminal exons of both genes located in close vicinity. The two genes were found to be arranged in opposite direction, head‐to‐head, separated only by a short region of 127 bp, apparently representing promoters of both genes as indicated by the existence of typical sequence motifs (CAT‐box, SP1 consensus sequence). These data suggest that the alpha 1(IV) and alpha 2(IV) genes use a common, bidirectional promoter. The striking symmetrical arrangement of sequence elements within the promoter may be of basic importance for the coordination of bidirectional transcription. The promoter region had no detectable transcriptional activity in transient gene expression assays after fusion to the chloramphenicol acetylase (CAT) gene in either direction, indicating the necessity of additional elements for efficient and tissue‐specific expression of both genes. Constructs containing different segments of both genes failed to identify regions with enhancing activity for the homologous collagen type IV promoter. When the heterologous HSV thymidine kinase promoter was used, a negatively acting region was identified. This indicates that the alpha 1(IV) and alpha 2(IV) promoter activity is controlled by additional regulatory elements present on distant portions of both genes.

The cDNA and protein sequences of the N-terminal6OY0 of the a2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oiigodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both a chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the a2(IV) chain as compared to the al(1V) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.

A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting >95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected >96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.As global population estimates reach 170 million infected with the hepatitis C virus (HCV) (23), there has never been a more pressing need for sensitive, precise tests for active infections. Although enzyme immunoassays (EIA) followed by confirmatory immunoblot assays have been traditionally used for screening and testing of blood, neither assay can differentiate between active and resolved infection. Qualitative and quantitative HCV RNA testing as well as HCV antigen detection methods can identify active infection, but with quantitative tests usually being 1 to 2 logs less sensitive than qualitative tests and with the limited availability of antigen methods, qualitative HCV RNA testing is the method of choice for confirming active infection and assessing viral clearance in response to therapy (8).Qualitative HCV RNA assays currently used are based on PCR technology and include the AMPLICOR HCV 2.0,

The genes COL4A1 and COL4A2, coding for the two subunit chains al(IV) and a2(IV) of collagen IV [al(IV) 2 (x2(IV)l are found closely linked on the human chromosome 13 in a unique head-to-head arrangement resulting in opposite strand transcription starting from a shared promoter region. Transient transfection experiments defined a shared promoter and two symmetrically arranged, downstream located and gene-specific activating elements in each gene. The shared promoter does not exhibit any transcriptional activity and efficient transcription depends on the cooperative effect of downstream elements. Mutual inhibitory effects between the two activating elements indicate competitive interactions with the shared promoter. Symmetry, cooperativity and competitivity of cis-elements are also reflected by the binding of transacting factors to the promoter and activating elements. From these data we propose a model for the coordination of divergent transcription of COL4 genes based on the cooperative and competitive interactions of the shared promoter and gene-specific regulating elements.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR‐T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR‐T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these “living” drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR‐T therapies. Herein, three main areas for monitoring CAR‐T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR‐T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit‐for‐purpose tools and help to more rapidly advance the development of next generation CAR‐T therapies.