Quantification of DNA using the luminescent oxygen channeling assay.

BACKGROUND: Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. METHODS: Luminescent oxygen channeling assay (LOCI(TM)) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. RESULTS: In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. CONCLUSIONS: This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

Cooperative and competitive interactions of regulatory elements are involved in the control of divergent transcription of human Col4A1 and Col4A2 genes.

The genes COL4A1 and COL4A2, coding for the two subunit chains alpha1(IV) and alpha2(IV) of collagen IV [alpha1(IV)2alpha2(IV)] 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.

Expression of human collagen type IV genes is regulated by transcriptional and post-transcriptional mechanisms.

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two alpha 1(IV) and one alpha 2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run-on analysis and mRNAs by RNAse protection assay indicates the involvement of transcriptional as well as post-transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady-state levels alpha 1(IV)/alpha 2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post-translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.

Regulation of divergent transcription of the genes coding for basement membrane type IV collagen.

The genes coding for the two polypeptide chains, alpha 1(IV) and alpha 2(IV), which form together the molecule of the basement membrane type IV collagen, were found to have a special and unusual genomic arrangement. The two genes are very closely linked, they are transcribed in opposite directions, and they apparently use a common and bidirectional promoter with a length of 127 bp. This region is characterized by a symmetrical arrangement of typical elements and by the palindromic structure of the sequence. In accordance with the symmetry of the promoter itself, a symmetrical organization of sequence motifs (SP1, CCAAT) was also observed in flanking regions. For the promoter and the flanking regions we could detect specific binding of nuclear factors that indicates their involvement in transcriptional activation. This suggests that the intrinsic symmetry of the type IV collagen promoter and its flanking regions may be a structural prerequisite for its bidirectional function. In transient gene expression systems no significant activity of the type IV collagen promoter was observed in either direction. This implies that additional enhancing elements are essential for the efficient and tissue-specific transcription of both type IV collagen genes. The screening for such controlling elements within the alpha 1(IV) and the alpha 2(IV) gene led to the observation that the transcription in direction of the alpha 2(IV) gene is activated by an element located in the first intron of the alpha 2 gene. Its enhancing effect is strictly dependent on the intact genomic structure of this region. Alternation of orientation and distance to the promoter destroys its activity completely. This element, located about 100-600 bp downstream from the start site of alpha 2(IV) transcription, seems to form a synergistically acting unit with the common promoter, essential for transcriptional activity in alpha 2 direction. We have not found additional enhancing elements in other regions of both genes. Explanations for the discrepancy with previous data, which define an enhancing element within the first intron of the alpha 1(IV) gene of mouse, are only speculative at present.

The genes for the alpha 1(IV) and alpha 2(IV) chains of human basement membrane collagen type IV are arranged head-to-head and separated by a bidirectional promoter of unique structure.

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.

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain.

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides 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 alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.