Improved internal control for molecular diagnosis assays.

The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. •The analyte and internal control have the same PCR and sequencing annealing sequences.•This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.•This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.

Identification of the severe acute respiratory syndrome coronavirus by simultaneous multigene DNA sequencing.

The recent severe acute respiratory syndrome (SARS) outbreak resulted in calls for an accurate diagnostic test that can be used not only for routine testing but also for generating nucleotide sequences to monitor the epidemic. Although the identity of the SARS coronavirus (SARS-CoV) genome was confirmed by DNA sequencing, it is impractical to sequence the entire 29-kb SARS-CoV genome on a routine basis. Therefore, alternative assay methods such as the enzyme-linked immunosorbent assay and PCR have been pursued for routine testing, primarily to resolve probable cases. We report here a modification of standard DNA sequencing technology for accurate identification of SARS-CoV in routine testing. Instead of requiring the sequencing of the whole SARS-CoV genome, our modification enables the simultaneous sequencing of three regions of the SARS-CoV genome, the spike protein-encoding gene (35 nucleotides), gene M (43 nucleotides), and gene N (45 nucleotides), in a single electropherogram. Comparing these nucleotide sequences to DNA databank entries (National Institutes of Health) conclusively identified them as SARS-CoV sequences.

Nucleotide sequence-based multitarget identification.

MULTIGEN technology (T. Vinayagamoorthy, U.S. patent 6,197,510, March 2001) is a modification of conventional sequencing technology that generates a single electropherogram consisting of short nucleotide sequences from a mixture of known DNA targets. The target sequences may be present on the same or different nucleic acid molecules. For example, when two DNA targets are sequenced, the first and second sequencing primers are annealed to their respective target sequences, and then a polymerase causes chain extension by the addition of new deoxyribose nucleotides. Since the electrophoretic separation depends on the relative molecular weights of the truncated molecules, the molecular weight of the second sequencing primer was specifically designed to be higher than the combined molecular weight of the first sequencing primer plus the molecular weight of the largest truncated molecule generated from the first target sequence. Thus, the series of truncated molecules produced by the second sequencing primer will have higher molecular weights than those produced by the first sequencing primer. Hence, the truncated molecules produced by these two sequencing primers can be effectively separated in a single lane by standard gel electrophoresis in a single electropherogram without any overlapping of the nucleotide sequences. By using sequencing primers with progressively higher molecular weights, multiple short DNA sequences from a variety of targets can be determined simultaneously. We describe here the basic concept of MULTIGEN technology and three applications: detection of sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum), detection of contaminants in meat samples (coliforms, fecal coliforms, and Escherichia coli O157:H7), and detection of single-nucleotide polymorphisms in the human N-acetyltransferase (NAT1) gene (S. Fronhoffs et al., Carcinogenesis 22:1405-1412, 2001).

Stress relief protein modulation by calnexin.

Oxygen free radical (OFR)-mediated oxidative stress in myocardial cells following ischemia could damage unit membrane and macromolecules such as nucleic acids (DNA). It is being reported that under this condition these cells produce antioxidants and heat shock proteins (HSP 70). It is implied that this family of proteins could function as a "molecular chaperone" in the cell and hence has to be transported to various target sites. This process is comparable to the induction of oxygen free radicals in melanocytes and its response, melanin production following UV light exposure stress. Lamp-1, trp-1 and tyrosinase are melanosomal-associated stress relief proteins which are involved in the production of melanin in the subcellular organelle, melanosomes. UV exposure studies as well as gene transfection studies and antisense hybridization in human melanoma cells clearly indicated an increase and marked coordinated interaction of all these stress relief proteins in melanogenesis. These proteins are synthesized in the endoplasmic reticulum and have to undergo posttranslation modification, sorting and posting to their respective target sites. We simultaneously identified and characterized an ER resident protein, calnexin. It became the potential candidate for "chaperoning" these proteins following translation. Based on the computer analysis of HSP 70 cDNA, we postulate that similar to stress response proteins in melanogenesis, stress relief proteins in myocardial cells may also be modulated by the same ER resident protein, calnexin.

A rapid non-selective method to generate quadromas by microelectrofusion.

A simple non-selective methodology was developed and standardized to generate desired hybrid-hybridoma or quadroma secreting bifunctional antibodies. This novel protocol is based on microelectrofusion on a meander chamber using a few hundred cells of each of the two parental hybridomas with no laborious drug selection procedures. Seeding approximately 10 cells per well in a 96-well microtitre plate after fusion in 200 microliters standard medium containing 20% FBS and 10% Origen growth factor generated positive quadromas secreting bispecific antibodies with good stability after the second reclone. Compared to the conventional PEG fusion and other methods this simple protocol is both rapid and economical. Generally, conventional methods to make quadromas and triomas require the introduction of drug selection markers into one or both of the parental cells, a procedure that could take 3-6 months. Utilizing the non-selective microelectrofusion method described here, we have generated several quadromas in a very short time. Further, such a protocol could also be potentially adopted to generate human hybridomas with few B cells isolated from peripheral blood lymphocytes enriched by antigen specific panning or affinity microelectrofusions.

Molecular control of melanogenesis in malignant melanoma: functional assessment of tyrosinase and lamp gene families by UV exposure and gene co-transfection, and cloning of a cDNA encoding calnexin, a possible melanogenesis "chaperone".

Melanogenesis is a cascade of events significantly controlled by regulatory genes which are associated with the melanosomal membrane. This report introduces our current research efforts dealing with (a) the gene and protein expressions of tyrosinase and Lamp (lysosome-associated membrane protein) families by human melanoma cells after repeated exposures to UV light, (b) the coordinated alterations in the expression of the Lamp family gene and its encoding product after transfection of two genes of the tyrosinase family in human melanoma cells and (c) cloning and sequencing of a Ca(2+)-binding phosphoprotein, calnexin, which could be a candidate as a chaperone for sorting and maturation of tyrosinase and Lamp family glycoproteins in melanogenesis cascade. Our UV exposure study, as well as gene transfection and antisense hybridization experiments, has clearly indicated a marked and coordinated interaction of the Lamp-1 gene with the tyrosinase and TRP-1 genes in this process. We propose that melanogenesis is controlled at least by two major gene family products, i.e., (a) the tyrosinase family of tyrosinase, TRP-1 and TRP-2, and the Lamp family of Lamp-1, Lamp-2 and Lamp-3. These two gene families probably derived from primordial melanogenesis-associated genes which are common or closely related to each other.

Identification of a cDNA coding for a Ca(2+)-binding phosphoprotein (p90), calnexin, on melanosomes in normal and malignant human melanocytes.

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca(2+)-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in lambda Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M(r) 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with beta-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca(2+)-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an antibody against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.

Characterization of melanosome-associated proteins by establishment of monoclonal antibodies and immunoscreening of a melanoma cDNA library through an anti-melanosome antibody.

Monoclonal antibodies against melanosomal components (human melanosome specific antigens; HMSAs) have been developed in our laboratory. HMSA-1-4 recognizes structural matrix proteins of melanosomes. HMSA-5 is identical to TRP-1, equivalent to the b (brown) locus of murine melanocytes and expressed in early stages of melanosomal maturation. HMSA-6 is a protein associated with melanosomes but its role is still unclarified, and HMSA-7 is identical to the lysosomal protein CD63. We have also recently identified p90 calnexin-like, Ca(2+)-binding protein p97 melanotransferrin, and p64 beta-D-galactosidase-like protein associated with melanosomes through immunological screening of our melanocytes (melanoma cells) cDNA library. Approximately 150 genes and 60 loci are known to influence eye, skin and hair colour in mammals. Tyrosinase is a rate-limiting enzyme responsible for melanin synthesis. In addition, tyrosine-related proteins (TRPs) and their genes have been identified and cloned. Tyrosinase and TRPS (e.g., TRP-1; b-locus protein identical to HMSA-5 and TRP-2; dopachrome tautomerase) are synthesized according to underlying genetic programmes, and are up- and/or down-regulated to create various forms of abnormal melanin pigmentation. We herein propose the importance of investigating the role of non-tyrosinase related proteins such as those which we have recently identified.

Molecular cloning, sequencing, and analysis of the cDNA for rabbit muscle glycogen debranching enzyme.

Six peptides were isolated from glycogen debranching enzyme purified from rabbit muscle, and their sequences were determined. A cDNA library made from rabbit muscle using random hexamer primers was screened with oligonucleotide probes constructed in accordance with these peptide sequences. Seven cDNA clones comprising the open reading frame were found, whereas oligo(dT) cDNA libraries yielded no positive clones because of the long 3'-nontranslated region of 2.3 kb. The open reading frame of 4665 bases codes for a 1555-amino-acid protein of M(r) 177,542. Compared to the sequence from human muscle, there are an additional 40 amino acid residues upstream from the N-terminus, and the next 10 residues show no homology. For the remaining 1505 residues, the two sequences exhibit an identity of 93%. The four consensus sequences commonly found at the carboxy termini of beta-strands in the alpha/beta barrel domains of amylases and glucanotransferases are also found in the N-terminal half of the debranching enzyme, suggesting that this structural domain may be present. This and other evidence suggests that the N-terminal half may encompass the transferase activity, leaving the glucosidase activity for the C-terminal half. The latter shows no significant homology to known proteins. An unusual feature of the sequence is the presence of three pairs of adjacent cysteines, which may explain inhibition of the enzyme by organic arsenites.

cDNA-based functional domains of a calnexin-like melanosomal protein, p90.

We have recently identified a gene encoding a calnexin-like protein (p90) by the immunoscreening of a human melanoma cDNA library, using a rabbit anti-human melanosomal antibody. This p90 protein was highly expressed by human melanocytes and associated with melanosomal membrane and endoplasmic reticulum. In this study we report the computer analysis of the predicted amino acid sequence of this calnexin-like melanosomal protein. We found that p90 is a membrane-bound protein whose large N-terminal domain is located within the melanosomal compartment; its shorter C-terminal is exposed to the cytosol and separated by a short transmembrane region. This p90 protein was found to have consensus sequences of a Ca(2+)-binding loop and a protein kinase C phosphorylation site at the N-terminal domain. The C-terminal domain, on the other hand, contained sequences of a casein kinase II phosphorylation site and two protein kinase A phosphorylation sites. Such functional motifs could provide signal transduction across the melanosomal membrane, the reception of melanogenic protein via carriers at the melanosomal membrane and the translocation of melanosomes in the melanocyte.

Novel type of plasmid-borne resistance to trimethoprim.

A novel trait for transferable resistance to high concentrations of trimethoprim was found to dominate among enterobacteria collected from different parts of Sri Lanka. Drug resistance was a result of the production of dihydrofolate reductase with a decreased sensitivity to antifolates. By characterization of the partially purified enzyme and by restriction enzyme digestion analysis, the newly found gene was shown to be distinct from the earlier known plasmid-borne resistance genes which express dihydrofolate reductases of types I, II, and III. Cloning of fragments containing the resistance gene and further restriction enzyme digestion analysis showed that this gene was inserted very close to a sulfonamide resistance gene. Evolution of trimethoprim resistance in Sri Lanka thus seems to have taken a different route from that taken in the industrialized world, where transposon Tn7 seems to dominate. The close combination of the new trimethoprim resistance gene with sulfonamide resistance on the plasmids studied would effect an efficient spread of these genes, since trimethoprim has most often been used in combination with a sulfonamide.

24-hour urine composition of Sri Lankan adults and their response to 100 mg dihydrochlorothiazide.

In the present study, 205 'normal hospitalised subjects' in the age group 16-60 years were included in a clinical trial to estimate the 24-hour urinary composition. Their response to 100 mg dihydrochlorothiazide was also determined. The study revealed that there is a marked difference in the 24-hour urine volume and composition between that observed in the present study and that presented in the modern literature; 100 mg dihydrochlorothiazide was found to produce a significant diuretic and natriuretic effect in normal Sri Lankan adults. There is a very high correlation between the volume of urine and the cations in the basal excretion as well as with the standard diuretic. The coefficient of regression of volume on cations in the basal excretion was found to be less than that with the standard diuretic.