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The study of molecular processes driving human neoplasia development is achieving critical gains in terms of better detection and treatment. Current medicine already benefits from discovering genetic signatures of distinct cancers, and therapies have become more specifically targeted to the molecular aberrations defining parti-cular cancers (e.g.: chronic myelogenous leukemia is now diagnosed by detecting the t(9;22)(q34;q11) translocation, and therapy is based on inhibitors of the aberrant bcr-abl kinase). Epigenetic processes complement the genetic determinants of the cellular phenotype, and their study is of great interest for cancer researchers. The primary physiologic role of epigenetics is to govern cellular differentiation. Epigenetics have particular importance in organogenesis and also in maintaining the proper phenotypic profile of each cell in distinct organs and systems. The vast majority of current studies address DNA methylation changes pertinent to different tumor types. The global hypomethylation occurring in cancer results in genetic instability and may trigger enhanced expression of particular oncogenes. In contrast, aberrant promoter hypermethylation may reduce the expression of proteins that are critical for tissue homeostasis. This mechanism may reduce the expression of important tumor suppressor genes or the level of proteins critically involved in DNA maintenance and repair, cellular adhesion and intracellular signaling. Apart from methylation, neoplasia related perturbations were identified in virtually all of the epigenetic machinery. Proteins controlling DNA conformation (such as histone– modifying enzymes, or components of ATP-dependent remodeling and poly-ADP ribosylation) show reduced expression or activity in several distinct tumor types, providing evidence that epigenetics are critically involved in cancer development. The dynamic profile of epigenetic changes provides valuable background for clinical applications. Based on the particular epigenetic signatures described for different tumors, current studies focus on the development of epigenetic tests not only to diagnose cancer in early stages including pre-invasive disease, but also to identify patients at risk for tumor development. In addition, neoplasia-related epigenetic changes open a new therapeutic approach in human cancers, with drugs that undergo or have already passed clinical trials. Epigenetics are therefore emerging as a promising field for both diagnostic and therapeutic approaches of human neoplasia and pre-invasive disease. [ABSTRACT FROM AUTHOR]

The human genome contains perhaps more than 50,000 genes. A typical cell in the human body is believed to express from 15,000 to 25,000 proteins. The majority of these proteins are expressed in all the cells, and are known as housekeeping genes. These are proteins involved in vital cell functions, including production of energy, oxidation, biosynthesis, regulation of cell cycle, and cell growth. The rest of the proteins are expressed differentially in various cell types. For example, some genes are expressed only in liver cells, muscle cells, or neurons, and not in any other cell types. These differentially expressed genes define the phenotype and function of a particular cell type. The identification of genes solely expressed in one cell type may shed light on mechanisms of cellular function and gene interaction. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor II (IGFII) growth control pathway is comprised of three major components: IGFII ligand, insulin-like growth factor I receptor (IGFIR), and insulin-like growth factor II receptor (IGFIIR). IGFIR and IGFIIR work together to maintain constant circulating levels of IGFII. IGFII is a potent mitogen, produced by epithelial cells, which stimulates cell growth and prevents apoptosis by binding to IGFIR (1,2). IGFIR is a member of the receptor tyrosine kinase family (3) Upon binding IGFII, IGFIR autophosphorylates tyrosine residues, thereby initiating signaling Fig. 1 ) (3). Both mitogenic and anti-apoptotic signaling pathways are initiated by the binding of IGFII to IGFIR (3). [ABSTRACT FROM AUTHOR]

Genomic fingerprinting is one of several methods for screening human genome to identify genetic alterations in cancer cells. Arbitrarily primed polymerase chain reaction (AP-PCR) is a PCR-based genomic fingerprinting method (1). In AP-PCR, a single oligonucleotide is used to initiate DNA synthesis from sites along the template with which it matches only imperfectly. PCR is performed under a low stringent condition in the initial five cycles to permit hybridization of arbitrary primers to various sequences dispersed throughout the human genome. This is followed by 30-35 cycles of PCR under high stringent condition, so that only the best matches of the initial annealing events are further amplified. By controlling the stringency of the initial cycles, 50-100 distinct DNA fragments can be amplified from the human genome by a single PCR. When those fragments are size fractionated by gel electrophoresis, a representative sample of cell genome is visualized by staining or autoradiography as a genomic fingerprint. Because of the arbitrary nature for designing a primer and the low stringent condition for hybridization of a primer, DNA fragments are simultaneously amplified from various chromosomal regions in a single PCR. By comparison of AP-PCR genomic fingerprints of DNA from tumors and normal tissues, deleted and amplified DNA sequences in cancer cells have been detected and cloned (2-5). Intensities of deleted and amplified DNA fragments are decreased and increased, respectively, by AP-PCR. [ABSTRACT FROM AUTHOR]

Patients suffering from Legionnaire's disease initially experience nonspecific symptoms such as fever, myalgia, and headache. The predominant clinical finding is pneumonia with symptoms ranging from a mild cough and slight fever to diffuse pulmonary infiltrates and multiorgan failure. The disease is caused by infection with Legionella, and since the outbreak of Legionnaire's disease in Philadelphia in 1976, resulting in 34 deaths among 221 cases (1), Legionella pneumophila has been established as an important cause of atypical pneumonia (2). [ABSTRACT FROM AUTHOR]

The advantages of direct sequencing of polymerase chain reaction (PCR) products over conventional sequencing of cloned, single-stranded DNA are manifold. Speed is perhaps the greatest asset. Time-consuming creation and screening of libraries, fragment purification, subcloning, bacterial transfection, and plasmid preparation steps are all eliminated. Cost is an advantage for similar reasons. The ability to sequence thousands or even millions of different templates at once, thereby obtaining pooled averages of mutated or polymorphic sequences, is another benefit. Moreover, short DNA sequences not obtainable by conventional cloning, such as DNA from paraffin-embedded tissues, can be sequenced using PCR sequencing. [ABSTRACT FROM AUTHOR]

Loss of heterozygosity (LOH) is being detected with increasing frequency in a wide variety of human tumors (1-10). Frequent LOH at a given chromosomal locus implies the existence of a tumor suppressor gene that is important in the pathogenesis of the particular cancer under study. LOH is believed to represent inactivation of one allele of a tumor suppressor gene by chromosomal or subchromosomal deletion. LOH studies have led to the isolation of new tumor suppressor genes (11-13). They have also pinpointed known tumor suppressor genes that may be significant in specific types of cancer (14,15). [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis, an obligate intracellular bacterium, and Trichomonas vaginalis, a protozoan, are two of the most prevalent sexually transmitted pathogenic microorganisms worldwide. C. trachomatis is a major cause of pelvic inflammatory disease, occluded fallopian tubes resulting in infertility, and ectopic pregnancy (1) whereas T. vaginalis frequently induces vaginitis, cervicitis, and urethritis (2). Both organisms are also associated with adverse pregnancy outcomes (3). [ABSTRACT FROM AUTHOR]

The rapid diagnosis of infectious diseases, particularly those because of their communicability present a public health problem, is the objective of the clinical microbiologist and the practicing physician. The need to attain this goal is exemplified by the resurgence of tuberculosis and the increasing prevalence of drug-resistant isolates of Mycobacterium tuberculosis. The recent introduction of molecular technologies for the laboratory diagnosis of infectious diseases into the clinical microbiology laboratory now makes the goal of truly rapid diagnosis of infectious diseases attainable. [ABSTRACT FROM AUTHOR]

Over the past decade, the genes that underlie the development of many human diseases have been identified and the diseases causing mutations within these genes have been unveiled. Many genetic alterations responsible for a variety of human disorders have been characterized. These alterations range from simple Mendelian inherited syndromes to more complex traits such as cancers that involve multiple genetic and environmental factors. Identification and characterization of disease-causing mutations has practical as well as biological implications. As our understanding of these alterations advances, the potential for developing molecular genetic markers with clinical applications increases. This improved understanding also opens new avenues for advances in diagnostic testing, prognostication, and design of preventative strategies or therapeutic interventions. Indeed, direct genetic testing for an inherited colorectal cancer predisposition syndromes, Familial Adenomatous Polyposis (FAP) is currently available to the medical community with appropriate genetic counseling (1). [ABSTRACT FROM AUTHOR]

The use of genetically altered mice in research has increased exponentially since the production of the first transgenic mouse 15 yr ago. Within the past decade, the technique of targeted mutagenesis in mice has seen a similar rapid expansion in use, becoming a strategy widespread throughout a number of laboratories studying a variety of experimental systems. In addition to the large number of new transgenic and knockout models being generated, many investigators are now performing combinatorial experiments in which various transgenic and/or knockout mice are intercrossed to produce animals with complex genotypes. These rapid experimental advances have necessitated the development of tools that permit efficient and precise identification of genetically altered alleles in mice. Many investigators use Southern blotting to identify transgenic founder mice or mice bearing homologous disruptions; however, this strategy often becomes laborious when large breeding programs are involved. PCR is a technique that lends itself very well to rapid identification of large numbers of mutant mice bearing complex genotypes. These large-scale analyses are often required when performing experiments with transgenic and knockout animals. [ABSTRACT FROM AUTHOR]

The approx 3×109 basepairs that comprise a human genome are believed to contain at least 100,000 different genes (1). Many of these genes and the proteins that they encode have been isolated and identified, but the vast majority of these genes have yet to be characterized. A typical mammalian cell is believed to express approx 15,000-25,000 different proteins. A significant percentage of these proteins are present in virtually all cell types and they are generally designated as "housekeeping genes." These include many of the various proteins involved in energy production, cellular biosynthesis, or the regulation of cell growth and division. The remainder of the proteins that the genome is capable of encoding are those that are differentially expressed. The expression of these proteins is often restricted to a few cell types or even just one particular cell type. For example, genes that are expressed only in a muscle or a kidney cell, or those that are found in a T or B lymphocyte, but not in both types of cells are differentially expressed. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most common malignancy diagnosed in men in the Western hemisphere. Once early tumors are detected, prognosis is largely unpredictable, and clinicians are currently unable to inform the patient whether his tumour is likely to progress. New criteria to define the aggressive and metastatic potential of prostate cancer are needed, particularly in view of the recent controversies and evidence from North American studies questioning the benefits of radical surgery over observation in early-stage prostatic adenocarci-noma (1-3). Furthermore, even in patients where the disease appears to be confined to the prostate, cancers can be understaged in over 50% of cases, with resulting positive surgical margins, extracapsular extension, and potential treatment failure (4). [ABSTRACT FROM AUTHOR]

Primary acquisition of Toxoplasma gondii (T. gondii) in healthy male and female adults is usually an asymptomatic infection (toxoplasma infection) or results in a harmless disease (toxoplasmosis) with nonspecific symptoms including headache, myalgia, fatigue, or a flu-like condition. In some cases, enlargement of lymph nodes in different body regions because of lymphatic spread of parasites together with slight fever might mimic infectious mononucleosis. [ABSTRACT FROM AUTHOR]

As a method of specific mRNA detection, the single most important advantage of RT-PCR is its sensitivity, because of the remarkable sensitivity of PCR. Isolation of polyA+ mRNA is unnecessary, and minute amounts of total RNA suffice. If random primers are used in the reverse transcription reaction, a single cDNA preparation can be used for the detection of numerous different mRNAs in the same RNA sample. This sensitivity means that RT-PCR is a powerful technique, particularly when tissue availability is limiting, or when the mRNA to be detected is present in low abundance. We have routinely detected low abundance mRNAs for various cytokine receptors in RNA samples isolated from small pinch biopsies of human colonic mucosal epithelium. The technique has also been used for the sensitive detection of viral RNA, such as from HIV and HCV, in human serum, where the viral titer is frequently <1000 virus particles per milliliter of serum. [ABSTRACT FROM AUTHOR]

The human hepatitis C virus (HCV) is an RNA virus discovered in 1989 that accounts for the majority of posttransfusion and sporadic non-A non-B hepatitis. More than half of patients with acute HCV infection develop a chronic course that is associated with a high risk of developing liver cirrhosis and hepa-tocellular carcinoma. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR) has proven to be an extremely powerful tool in the field of virus detection. It has been employed in a variety of situations where previous techniques were too slow, too cumbersome, or simply nonexistent. Some of these are as follows. [ABSTRACT FROM AUTHOR]

Genetic transformation of plants has enormously matured within the last few years. It began with the introduction of genes that imparted resistance to cultivated plants against herbicides and pathogenes. More recently, there are approaches to modify the quality of plants, such that useful traits are amplified, and/or unwanted traits are eliminated. Of particular interest in medical research are experiments in which genes are introduced into the plant genome that allow expression of herbal drugs and antibodies. [ABSTRACT FROM AUTHOR]

The identification of mutations is very important in such aspects of molecular biology as medical diagnostics, ascertaining structure/function relationships, population genetic studies, and in confirming the authenticity of new candidate genes. Presently there are a variety of different techniques used to identify somatic and germline mutations. In some instances, gross genetic alterations are best characterized by cytogenetic analysis, FISH, or by Southern blot. However, in most cases the underlying mutations are too subtle to be revealed by these techniques and are best characterized by examination of PCR products generated from putative disease alleles. While there are over a dozen different methods to screen for mutant alleles in PCR products, no technique is absolutely sensitive and each has specific advantages and disadvantages in its ability to detect unknown mutations. [ABSTRACT FROM AUTHOR]

In addition to the Gag, Pol, and Env structural proteins, HIV-1 encodes at least six regulatory proteins: Tat, Rev, Nef, Vif, Vpr, and Vpu. All HIV-I proteins are encoded by overlapping reading frames and are expressed through the complex alternative splicing of a single precursor RNA leading to three major RNA classes (8-8): an unspliced class which includes both genomic RNA and gag-pol mRNA; a singly spliced class that includes mRNAs coding for Env, Vpu, Vif, Vpr, and a truncated form of Tat protein; and a multiply-spliced class that includes mRNAs coding for the regulatory proteins Tat, Rev, and Nef (Fig. 1). The function of the regulatory proteins Tat, Rev, Nef, Vif, Vpr, and Vpu has not yet been fully elucidated. Nevertheless, they seem to play specific roles during the different steps of the HIV-1 replication cycle (9-11). For this reason, the detection of mRNA species encoding these proteins at different steps of the virus replication cycle will provide important information about the function of these proteins. In order to obtain both a quantitative and qualitative detection of these mRNAs, we used a ribonuclease protection assay. Ribonuclease protection assays are commonly used for the detection and quantification of mRNAs (12,13) and are well-suited for mapping the position of internal and external junctions in mRNAs (14,15). In addition, as the hybridization takes place in liquid conditions, this technique is more sensitive than other quantitative methods of detection of RNAs such as Northern blotting (16). Our assay uses a DNA template specific of the HIV-1 strain to be investigated. [ABSTRACT FROM AUTHOR]

Fingerprinting techniques are essential tools in the investigation of transmissible diseases. Unless one is able to track a pathogenic organism from its reservoir, through its vectors and carriers into infected hosts, it is impossible to define an organism's epidemiology. In the absence of such understanding public health and other measures directed toward the eradication of the infection are unlikely to succeed. [ABSTRACT FROM AUTHOR]

World-wide enteric pathogens are a leading cause of potentially preventable morbidity and mortality. They are responsible for an estimated 700-1000 million episodes of diarrhea and cause four to five million deaths each year (1). Infants and young children in the developing world are particularly prone to infection and dehydration. Escherichia coli was identified as the causative agent with four main groups recognized as both animal and human enteropathogens; enterohemorrhagic E. coli (EHEC) enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). [ABSTRACT FROM AUTHOR]

In 1987, a disease syndrome characterized by reproductive and respiratory symptoms in pigs was first described in the United States (1). Late-term abortions, increased numbers of stillborn and weak pigs, poor conception in breeding herds, along with respiratory distress and high mortality in suckling, weaned, and grow-finish pigs was observed (2,3). Similar outbreaks were also described in Europe in 1990 (4). In 1991, the etiologic agent was identified as an enveloped, single-stranded RNA (ssRNA) virus and tentatively classified in the Arteriviridae family, which includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus (2,5,6). The porcine reproductive and respiratory syndrome virus (PRRSV) or Lelystad virus (the European PRRSV isolate) has a genome comprised of seven open reading frames (ORFs). ORFs 1a and 1b; ORFs 2-6 and ORF 7 are predicted to encode for the RNA polymerase, viral membrane associated proteins, and nucleocapsid, respectively (7). [ABSTRACT FROM AUTHOR]

Since the polymerase chain reaction (PCR) for DNA amplification was first introduced in 1985 (1), the combination of reverse transcription with subsequent PCR amplification of the cDNA (RT-PCR) has been an increasingly utilized technique to analyze gene expression (2,3). In order for this procedure to be reasonably quantitative, however, appropriate controls must be applied to all steps, including the quantitation of the original RNA, the reverse transcription, and the PCR itself. Several investigators have published methods on quantitative RT-PCR that involve varying cDNA input into the PCR (3-7), varying cycle number (3,4,8,9), or the use of a competitive template as an internal standard (10,11). However, only a few of the competitive PCR methods take into account the efficency of the reverse transcription phase of RT-PCR (4,7,11), which may vary from 10-50% (12,13). In the former methods, it would also be necessary to amplify another control gene in parallel (e.g., actin) to control for RNA input and reverse transcription. [ABSTRACT FROM AUTHOR]

The bovine immunodeficiency-like virus (BIV), also known as the bovine lentivirus or the bovine immunodeficiency virus, causes a persistent viral infection of cattle, and is found in cattle populations around the world (1,2). A closely related lentivirus, Jembrana disease virus, causes an acute disease in Bos javanicus cattle, and while it also infects Bos taurus and Bos indicus cattle the infection is generally asymptomatic (3). Strong evidence has not been presented that links BIV to a specific cattle disease, but BIV has been associated with decreased milk yield and with a variety of disease conditions including clinical immunodeficiency, encephalitis, bovine paraplegic syndrome, skin infections, and emaciation (4-6). [ABSTRACT FROM AUTHOR]

The Marfan syndrome (MFS) is an autosomal dominant heritable connective tissue disorder characterized by variable and pleiotropic manifestations primarily in the skeletal, ocular, and cardiovascular systems (1). Molecular defects in fibrillin-1, a 350 kDa glycoprotein, encoded by the FBN1 gene located on chromosome 15 are now well documented in individuals and families with MFS (seeref. 2 for a review). The variability in phenotypic expression of MFS is significant not only between families, but also within a family, while the effective treatment of patients with MFS relies heavily on early and accurate diagnosis. It, therefore, seems likely that prenatal and presymptomatic molecular genetic analysis would prove a valuable adjunct to clinical diagnosis for purposes of risk assessment (3-5). Mutations have been found along the entire 10 kb coding region of fibrillin and in most of its 65 exons. In addition, most families appear to have unique mutations. Therefore, the routine use of mutation screening for prenatal and presymptomatic diagnosis is impractical in this disorder. We have used intragenic polymorphic markers in FBN-1 (4) to perform linkage analyses in MFS families. We have adapted a "one-step" or "two-step" fluorescence PCR and automated sequencer to accurately, reproducibly, and rapidly perform prenatal or presymptomatic diagnosis of MFS in informative families. [ABSTRACT FROM AUTHOR]

Since the discovery of the doublehelix structure of DNA (1), no single event has had the same impact on the field of molecular biology as the rediscovery by Kary Mullis in the early 1980s of the polymerase chain reaction (PCR) (2-4), which was first published in principle by Keld Kleppe in 1971 (5). This elegant technology with its apparent simple theory has revolutionized almost every aspect of classical molecular biology, and is at the present moment beginning to make a major impact upon many medical—especially diagnostic-specialities. The field of clinical microbiology has been among the first to embrace the polymerase chain reaction technology, and the expectations of the future impact of this technology are high. First and foremost, the diagnostic possibilities of this technology are stunning, but in this era of emerging implementation, it is crucial to focus not only on the possibilities, but also on the pitfalls of the technology. Failure to do so will increase the cost of implementation manifold, and will risk to disrepute the technology in the eyes of the clinicians. [ABSTRACT FROM AUTHOR]

Selective ultraviolet radiation fractionation (SURF) is a simple technique for the isolation of histologically defined microscopic tissue regions (1,2). Very small numbers (100-400) of cells can be rapidly isolated with relatively crude equipment. The isolated cells can be analyzed genetically by PCR, thereby allowing a direct comparison between microscopic phenotype with genotype. The ability to compare genotype between different tissue areas provides opportunities to analyze many disease processes, including the heterogeneity expected of multistep tumor progression. [ABSTRACT FROM AUTHOR]

The rodent parvoviruses include mouse parvovirus (MPV), minute virus of mice (MVM), H-1 parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus (RPV), and hamster parvovirus (HaPV). Recent reports suggest LuIII, an autonomous parvovirus of unknown host origin, may also belong to the rodent parvovirus group (1,2). In general, rodent parvoviruses cause subclinical infections in their host of origin, although naturally occurring clinical disease may occasionally occur secondary to KRV or HaPV infection (3,4). Probably of more importance to the scientific community are the potential adverse effects these agents may have on research that utilizes rodents with inapparent and undetected infections. Subclinical rodent parvovirus infections may alter the immune response or cause fetal resorption or oncosuppression (5). In addition, rodent parvoviruses can cause persistent infections in cell culture and transplantable tumor lines (6,7), and therefore can adversely affect experiments performed in vitro. [ABSTRACT FROM AUTHOR]

Radioactively labeled polymerase chain reaction (PCR) products are being used in an increasing number of molecular biology research techniques. Among these are PCR-based polymorphism assays such as linkage analysis (1) and detecting allelic loss in cancer cells (2). Other uses of radioactive PCR include generating probes for Southern and Northern blotting (3) and screening for polymorphisms and point mutations by the single-strand conformation polymorphism (SSCP) technique (4). [ABSTRACT FROM AUTHOR]