Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site.

DNA molecular biology in the diagnosis of pulmonary disease.

DNA molecular biology is becoming increasingly important in clinical medicine. It has provided a new method to diagnose an inherited pulmonary disease in which the biochemical defect has been defined. Alpha-1-antitrypsin deficiency can now be diagnosed by direct analysis for the disease gene. In cystic fibrosis, another inherited pulmonary disease, the biochemical defect has not yet been defined. However, the search has been narrowed. The cystic fibrosis genetic defect has recently been localized to the long arm of chromosome 7. Because polymorphic DNA markers (RFLPs) are available for this region of chromosome 7, it is now often possible, using linkage analysis, to trace the inheritance of the cystic fibrosis gene(s) in families known to have cystic fibrosis. When the cystic fibrosis genetic defect is defined, it will then be possible to look directly for the disease gene. Finally, the greatest impact in clinical medicine may well be the development of rapid DNA hybridization techniques to diagnose infectious diseases that currently take days to weeks to diagnose.

Sequences which flank an 11p deletion observed in an hepatocellular carcinoma map to 11p13.

There is considerable interest in the 11p13 region because of its involvement in Wilms tumor, sporadic aniridia, and other congenital abnormalities. Cloned DNA sequences from this region might be useful in understanding the chromosomal abnormalities which lead to such disorders. However, few such markers exist. Using somatic cell hybrids which contain defined 11p deletions, two cloned DNA sequences which flank a deletion generated in an hepatocellular carcinoma (as a consequence of hepatitis B virus integration) were mapped to 11p13. Thus both ends of the deletion observed in an hepatocellular carcinoma are within 11p13.

Small cell lung cancer.

Lung cancer is the predominant fatal neoplasm of our time, and SCLC, which accounts for about 25% of all lung cancer, if untreated results in death in about 3 months. Currently employed aggressive combination chemotherapy has allowed a 4- to 5-fold improvement in median survival over untreated patients. Ten to 20% of patients with limited disease can be expected to have a long-term (2-yr) survival. The majority of patients, however, have extensive disease. For these patients the median survival is about 7 months. Less than 2% survive 2 yr. During the last 10 yr, experience in the treatment of thousands of patients has been reported. These trials, using a large variety of drug combinations, doses, and schedules as well as multiple modalities including radiotherapy, surgery, and bone marrow transplantation, demonstrate that a plateau has been reached with our present therapeutic approach. The development of new effective therapeutic strategies as well as prevention of SCLC require a better basic understanding of the cellular pathophysiology of the disease. A consistent chromosomal abnormality has been associated with SCLC. This may provide new insight into predisposition and pathogenesis of SCLC. How this chromosomal abnormality relates to loss of control of cell growth is under intense investigation. Similarly, during the past 3 yr, the identification of growth regulatory oncogenes has greatly improved our understanding of malignancy. The discovery that metastatic cells escape immune surveillance has led to attempts at modulating antigenic expression. The modulation of cellular antigenic expression may facilitate the destruction of tumor cells by host defense mechanisms. The understanding of the genetic basis of drug resistance may lead to approaches that prevent or delay resistance. This century has witnessed the emergence of SCLC as an important fatal neoplasm. It has also been during this time that another, formerly dominant pulmonary condition, tuberculosis, has been controlled. The reduction of tuberculosis was accomplished by a combination of scientific understanding, beginning with the discovery of Koch's bacillus, and public health measures. Perhaps a similar parallel for SCLC as well as other forms of cancer will be written. Basic cellular investigations with the new tools of molecular biology as well as measures to control exposure to predisposing environmental factors such as component of cigarette smoke may one day lead to control of SCLC.

Restriction fragment analysis of chromosomal DNA defines different strains of Mycobacterium tuberculosis.

As an initial step in gaining a better understanding of the important clinical properties that vary between strains of mycobacteria, we attempted to find molecular markers that would define different strains of Mycobacterium tuberculosis. We used restriction fragment analysis with the endonuclease MboI and hybridization with total M. tuberculosis DNA to examine DNA differences between 15 strains of M. tuberculosis. We were able to identify different strains using this method. In order to assess the sensitivity of this method in identifying different strains, we compared it with phage typing. The 2 methods appear to be similar in sensitivity and also to be complementary. There were 2 examples where restriction fragment analysis did not separate strains with different phage types. In addition, there were 2 examples where phage typing did not separate strains with different restriction patterns. Finally, there were 2 epidemiologically unrelated strains with the same restriction pattern and the same phage type. This method of restriction fragment analysis of chromosomal DNA is potentially useful for epidemiologic studies of tuberculosis. Additionally, by analyzing the genome of M. tuberculosis, molecular markers may well be defined that will be useful in discovering the pathogenesis of the clinical properties of M. tuberculosis, which previously have been poorly understood.

Regional assignment of the polymorphic probe D3S3 to 3p14 by molecular hybridization.

The polymorphic DNA probe D3S3 was assigned to 3p14 by molecular hybridization using a human chromosome 3/hamster somatic cell hybrid deletion panel. This is the first regional assignment of a polymorphic probe to the short arm of chromosome 3. This probe appears to be proximal to the chromosome 3 fragile site and, therefore, may prove useful in characterizing the 3p chromosomal aberrations that occur in various malignant diseases.

The new biomedical technology.

New methods for studying the genetic information of humans in health and disease are emerging from basic science laboratories. Because these approaches are yielding fundamental insights for diagnosing and treating disease, it is important that practitioners begin to understand these methods and how they are used. Methods for genetic analysis using recombinant DNA techniques consist of isolation, separation, propagation in microorganisms and molecular hybridization of DNA. The study of RNA allows determination of gene expression. These methods are being used to understand cancer, identify hereditary illness, produce pharmaceuticals and diagnose common clinical problems, such as infectious diseases.

The E7-associated cell-surface antigen: a marker for the 11p13 chromosomal deletion associated with aniridia-Wilms tumor.

Unbalanced interstitial deletions of the p13 region of human chromosome 11 have been associated with congenital hypoplasia or aplasia of the iris, mental retardation, ambiguous genitalia, and predisposition to Wilms tumor of the kidney. Utilizing somatic cell hybrids containing either the normal or abnormal chromosome 11 from a child with Wilms tumor and aniridia, we previously mapped the E7 cell-surface antigen to the 11p1300-to-11p15.1 region. To localize even further the site of this antigen on chromosome arm 11p, we have produced somatic cell hybrids from the fibroblasts of a second child with Wilms tumor and aniridia and a different deletion of 11p [46,XY, del (11)(pter----p14.1::p11.2----qter)]. Furthermore, the normal and deleted chromosome 11 could also be distinguished on the basis of a restriction fragment length polymorphism for the beta-globin gene. Hybrid cells containing the deleted chromosome were not killed in the presence of complement and the E7 monoclonal antibody (which recognizes E7 cell surface antigen), while hybrid cells containing the patient's normal chromosome 11 were killed. Thus, expression of the E7-associated cell-surface antigen can be mapped to the 11p13 region, and it appears to be a potential marker of the chromosome abnormality associated with aniridia-Wilms tumor.

Techniques of DNA hybridization detect small numbers of mycobacteria with no cross-hybridization with non-mycobacterial respiratory organisms.

The traditional methods used in identifying mycobacteria, such as acid-fast bacillus stains and culture, are often time-consuming, insensitive, and nonspecific. As part of an ongoing program to improve diagnosis and characterization of mycobacteria, we have found that deoxyribonucleic acid (DNA) hybridization techniques using isotopically labeled, single-stranded, total DNA can be used to detect as little as 10(-4) micrograms of Mycobacterium tuberculosis (MTb) DNA. This amount of DNA represents approximately 2 X 10(4) genomes. We have also shown the MTb DNA is sufficiently different from the DNA of non-mycobacterial microorganisms such that cross-hybridization with MTb DNA does not occur under the hybridization conditions we employed. We speculate that DNA hybridization techniques may allow the rapid, sensitive, and specific identification of mycobacteria.

A method of isolating nick-translated DNA by subsequent separation on low-melting-temperature agarose.

The use of nick translation of cloned DNA segments followed by separation on low-melting-temperature agarose has been used to obtain multiple radiolabeled DNA probes from a single nick-translation procedure. This technique avoids gel matrix inhibition of enzymatic reactions. Examples of the utility of this procedure are presented and the advantages and drawbacks discussed.

Cystic fibrosis. A disorder of adults too!

Cystic fibrosis is the most frequently occurring lethal hereditary disorder among whites. Many persons with cystic fibrosis now live into their adult years, and some cases are not diagnosed until adulthood. As a result, the disorder is no longer rare in adults. Respiratory symptoms and findings usually predominate, but a host of other complications that arise in adults also present therapeutic challenges to physicians. While no cure is yet available for cystic fibrosis, genetic counseling can help couples determine their relative risk for parenting a child with the disorder.

Wilms' tumor-aniridia association: segregation of affected chromosome in somatic cell hybrids, identification of cell surface antigen associated with deleted area, and regional mapping of c-Ha-ras-1 oncogene, insulin gene, and beta-globin gene.

Fusion of an auxotrophic mutant hamster cell with the skin fibroblasts of a child with the Wilms' tumor-aniridia association produced clones which, on the one hand, contained the child's normal chromosome 11 and, on the other, the chromosome 11 with the 11p13 deletion associated with the syndrome. Both hybrids were positive for human LDH-A by enzymatic assay. Clones containing the normal human chromosome 11 were killed by a cytotoxic monoclonal antibody to a cell surface antigen previously mapped to the 11p13----11pter region of chromosome 11. Clones with the abnormal 11 were not killed. Thus, we have produced hybrids from the same patient distinct from each other on the basis of their chromosome 11. These hybrids have been used to map the locus for a cell surface antigen to the deleted region on chromosome 11 of a patient with the Wilms tumor-aniridia association. The linkage between this antigen and the syndrome should be helpful in further study of the genetics of this disease. In addition, we have found that the c-Ha-ras-1 oncogene is distal to the p13 region of chromosome 11 and the position of insulin and beta-globin on the chromosome. Finally, by producing segregants of the hybrids containing the abnormal chromosome 11, we have provided evidence that chromosome 11-associated c-Ha-ras-1 is syntenic with chromosome 11 and not moved to a different portion of the genome.

Bitolterol compared to isoproterenol in advanced chronic obstructive pulmonary disease.

Twenty-eight patients with advanced emphysema and/or chronic bronchitis and severe airflow obstruction were randomly assigned to receive either bitolterol or isoproterenol aerosol delivered by a metered dose device which was administered three times daily. Randomization resulted in similar patients with like degrees of airflow obstruction and responsiveness to a test dose of inhaled bronchodilator. Significantly greater improvement in airflow was achieved by administration of bitolterol compared to isoproterenol. Pharmacologic responses continued after 90 days of daily dosing. Both drugs were well tolerated and side effects included mild degrees of tachycardia for both drugs. Two patients assigned to isoproterenol stopped therapy during the study due to side effects. This study indicates that bitolterol is more effective than isoproterenol in degree and duration of bronchodilatation in patients with advanced chronic obstructive pulmonary disease.

Molecular hybridization under conditions of high stringency permits cloned DNA segments containing reiterated DNA sequences to be assigned to specific chromosomal locations.

Identifying the specific DNA sequences involved in the chromosomal abnormalities in developmental and neoplastic diseases may be essential to understanding the molecular biology of these disorders. The use of recombinant DNA techniques in conjunction with rodent-human hybrid cells makes it possible to assign chromosomal locations to specific DNA sequences. However, the ubiquitous presence of reiterated DNA species often complicates the application of straightforward molecular hybridization. To accelerate the mapping of cloned sequences to specific chromosomal locations, we investigated the possibility that cloned sequences containing reiterated DNA might be used without isolating unique sequences. By varying conditions of hybridization (specifically temperature) and using restricted DNA samples from human genomic DNA, Chinese hamster ovary-human chromosome 11 hybrids, and non-chromosome 11 hybrids, we have been able to assign cloned DNA sequences containing reiterated sequences to their chromosome of origin. By hybridization under the high-stringency condition of 55 degrees C, specific banding was produced with both human genomic DNA and the human-chromosome-containing hybrid from which the probe was prepared. Furthermore, using a panel of chromosome 11 deletion mutants, we have been able to assign a cloned sequence to a specific chromosomal location. We believe that this approach will accelerate gene mapping procedures and facilitate identification of DNA sequences involved in chromosomal abnormalities.

Aerosolized terbutaline sulfate in the treatment of bronchospasm.

Terbutaline sulfate, a relatively selective beta2 agonist, is indicated for the treatment of bronchospasm associated with chronic obstructive pulmonary disease. Studies have shown that when administered via an aerosol, terbutaline has a rapid onset, a prolonged duration of action, and a low incidence of systemic side effects. Because the drug is delivered directly to the bronchi, one can administer low doses of aerosolized terbutaline and achieve a bronchodilatory effect comparable to that achieved with higher doses of the oral form.

Factitious illness. Dramatic deceit versus reality.

Patients with factitious illness present a particular challenge. Because they may be clever in their deception, they may be difficult to recognize as the fabricators of their apparent medical problem(s). Findings vary from relatively simple problems such as fever to more complex and often dramatic complaints of bleeding and pain. Differential diagnosis should include other, often more easily managed disorders such as somatoform disorders, malingering, antisocial personality, and schizophrenia. Because no definitive treatment exists, patients often consult many physicians, often in diverse geographical locations.