ABSTRACT
Thirty-nine patients with condylomas (12 women and 27 men) attending a dermatology clinic were tested for genital human papillomavirus (HPV) DNA and for seroprevalence to HPV type 6 (HPV6) L1 virus-like particles. The L1 consensus PCR system (with primers MY09 and MY11) was used to determine the presence and types of HPV in sample specimens. All 37 (100%) patients with sufficient DNA specimens were positive for HPV DNA, and 35 (94%) had HPV6 DNA detected at the wart site. Three patients (8%) had HPV11 detected at the wart site, and one patient had both HPV6 and -11 detected at the wart site. Thirteen additional HPV types were detected among the patients; the most frequent were HPV54 (8%) and HPV58 (8%). Baculovirus-expressed HPV6 L1 virus-like particles were used in enzyme-linked immunosorbent assays to determine seroprevalence among the patients with warts. Seronegativity was defined by a control group of 21 women who were consistently PCR negative for HPV DNA. Seroprevalence was also determined for reference groups that included cytologically normal women who had detectable DNA from either HPV6 or HPV16 and women with HPV16-associated cervical intraepithelial neoplasia. Among the asymptomatic women with HPV6, only 2 of 9 (22%) were seropositive, compared with 12 of 12 (100%) female patients with warts. A similar trend in increased HPV6 seropositivity with increased grade of disease was found with the HPV16 DNA-positive women, whose seroprevalence increased from 1 in 11 (9%) in cytologically normal women to 6 in 15 (40%) among women with cervical intraepithelial neoplasia 1 or 3. However, only 4 of 25 (16%) male patients were seropositive. No factors examined, such as age, sexual behavior, or a history of warts, were found to definitively account for the gender difference in seroresponse.
Subject(s)
Condylomata Acuminata/virology , Papillomaviridae/classification , Antibodies, Viral/blood , Case-Control Studies , Cervix Uteri/virology , Cohort Studies , Condylomata Acuminata/immunology , DNA Probes, HPV , Female , Humans , Inclusion Bodies, Viral/ultrastructure , Male , Microscopy, Electron , Papillomaviridae/immunology , Papillomaviridae/ultrastructure , Polymerase Chain Reaction , Vulva/virologyABSTRACT
A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.
Subject(s)
DNA Mutational Analysis , Membranes, Artificial , Oligonucleotide Probes , Base Sequence , Cell Line , Cystic Fibrosis/genetics , DNA , Genes, ras , Genetic Diseases, Inborn/genetics , Humans , Molecular Sequence Data , Neoplasms/genetics , Nucleic Acid Hybridization , Oligonucleotide Probes/chemical synthesis , Polymerase Chain Reaction , Surface PropertiesABSTRACT
The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, we have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.
Subject(s)
Leukemia, Lymphoid/diagnosis , Leukemia, Myeloid/diagnosis , Philadelphia Chromosome , RNA, Messenger/analysis , RNA, Neoplasm/analysis , Cell Line , Gene Amplification , Humans , Immunoassay , Karyotyping , Leukemia, Lymphoid/genetics , Leukemia, Myeloid/genetics , Nucleic Acid Hybridization , RNA, Messenger/immunologyABSTRACT
Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.
