Biomedical subjects

J W Yoon

Publications and source records attributed to J W Yoon.

At least 91 records · Page 5Linked to original sources

PubMed1991

Studies on autoimmunity for initiation of beta-cell destruction. VII. Evidence for antigenic changes on beta-cells leading to autoimmune destruction of beta-cells in BB rats.

The diabetic syndrome in BioBreeding (BB) rats is believed to result from the destruction of beta-cells by autoimmune responses. However, the initial events that cause the autoimmune destruction of beta-cells remain largely unknown. This investigation was initiated to see whether there are any antigenic changes on the beta-cells from neonatal to adult BB rats that may lead to the autoimmune destruction of beta-cells. Pancreatic grafts from neonatal BB rats remained largely intact without insulitis when transplanted into the renal subcapsular space of acutely diabetic BB rats. Similarly transplanted islet grafts from neonatal BB rats were also not subject to autoimmune destruction. In contrast, islet grafts obtained from adult BB rats, which had been treated with silica to prevent insulitis, were rapidly destroyed in diabetic recipients. These results indicate that beta-cells from neonatal BB rats are different from beta-cells from adult BB rats, at least regarding their recognition by immunologic effectors. Considering our observations and previous information on the initial role of macrophages/dendritic cells in the development of insulitis in BB rats, we suggest that beta-cell-specific antigenic changes that precede insulitis may result in the autoimmune destruction of beta-cells in BB rats.

Aging↗

PubMed1991

Role of viruses in the pathogenesis of IDDM.

Insulin-dependent diabetes mellitus (IDDM), also known as type I diabetes, results from the destruction of pancreatic beta cells. During the past few decades, genetic factors, autoimmunity and viral infections have been extensively studied as the possible cause of beta cell destruction. The evidence for virus-induced diabetes comes largely from experiments in animals, but several studies in humans also point to viruses as a trigger of this disease in some cases. There are at least two possible mechanisms for the involvement of viruses in the pathogenesis of IDDM: (a) cytolytic infection of beta cells may result in destruction of the cells without the induction of autoimmunity, or may be a final insult leading to the clinical onset of diabetes in individuals with an already decreased beta cell mass resulting from an autoimmune process; and (b) persistent viral infection (e.g. retrovirus, rubella virus, cytomegalovirus) may result in the triggering of autoimmune IDDM in certain circumstances. Regarding the latter possibility, viruses may insert, expose, or alter antigens in the plasma membrane of the beta cell, which may initiate autoimmunity leading to the destruction of the cells.

Animals↗

PubMed1990

The role of viruses and environmental factors in the induction of diabetes.

The development of IDDM results from the destruction of pancreatic beta cells. Genetic factors, various immune system alterations, and environmental factors have been studied as the possible causes of IDDM. The concordance rate for developing IDDM between monozygotic twins approaches 50%, suggesting that genetic factors are necessary, but nongenetic factors such as various immune system alterations and environmental factors also influence the clinical expression of genetic susceptibility. Environmental factors (e.g., viruses, chemicals, and diet) affecting the induction of diabetes may act as primary injurious agents which damage pancreatic beta cells or as triggering agents of autoimmunity. Certain viruses including EMC-D and Mengo virus 2T can directly infect pancreatic beta cells and replicate in the cells. The replication of viruses in the beta cells results in the destruction of the cells within 3 days, and the infected mice develop a diabeteslike syndrome in 3-4 days without the involvement of autoimmunity. In contrast, rubella virus appears to be somewhat weakly associated with autoimmune IDDM in hamsters. In addition, endogenous retrovirus expressed in pancreatic beta cells is clearly associated with the development of insulitis and diabetes in NOD mice. In man, there appears to be no correlation between the detection of islet cell autoantibodies and anti-Coxsackie B viral antibodies in newly diagnosed IDDM. In contrast, persistent infection of CMV and rubella virus appears to be associated with the presence of autoantibodies in newly diagnosed IDDM patients. It is particularly noteworthy that human CMV can induce islet cell autoantibodies that react specifically with a 38 kDa islet cell protein which may represent islet cell-specific antigens in a proportion of CMV-associated IDDM cases. These observations suggest that the association of diabetes with Coxsackie B viruses might be due to cytolytic infection of the beta cells with no link to autoimmunity, while both rubella virus and CMV are probably associated with autoimmune IDDM. A number of structurally diverse chemicals including alloxan, streptozotocin, chlorozotocin, Vacor, and cyproheptadine are diabetogenic mainly in rodents and sometimes in man. Possible mechanisms for beta cell destruction by these chemicals include (a) generation of oxygen free radicals and alteration of endogenous scavengers of these reactive species; (b) breakage of DNA and a consequent increase in the activity of poly-ADP-ribose synthetase, an enzyme depleting nicotinamide adenine dinucleotide in beta cells; and (c) inhibition of active calcium transport and calmodulin-activated protein kinase activity. (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Predisposing effect of anti-beta cell autoimmune process in NOD mice on the induction of diabetes by environmental insults.

In NOD mice. 50-70% of females and 10-20% of males develop diabetes, although almost all the animals show insulitis. To see if environmental insults could induce diabetes in subjects with pre-clinical anti-Beta cell autoimmunity, non-diabetic NOD mice were selected and injected with a sub-diabetogenic dose of streptozotocin at 6 or 20 weeks of age. The streptozotocin failed to induce diabetes in 16 male and 16 female NOD mice within 4 weeks when they were injected at the age of 6 weeks. In contrast, 6 of 16 male and 10 of 16 female NOD mice developed diabetes within 4 weeks when they were injected at the age of 20 weeks. In untreated age-matched control NOD mice, none of the male and only 2 of 16 female mice became diabetic during the same 4 week period. On histologic examination, the degree of insulitis in streptozotocin-treated NOD mice (at the age of 24 weeks) was not significantly different from that of untreated control NOD mice. However, the streptozotocin-treated animals showed significantly lower pancreatic insulin content than the control mice. These results show that an anti-Beta cell autoimmune process in NOD mice has a predisposing effect on the induction of diabetes by a sub-diabetogenic dose of streptozotocin, and suggest that the precipitation of clinical diabetes by some environmental insults in subjects with pre-existing pre-clinical autoimmune Beta-cell destruction may be one mechanism of disease presentation in human Type 1 (insulin-dependent) diabetes.

Animals↗

PubMed1990

Human pancreatic islet cell specific 38 kilodalton autoantigen identified by cytomegalovirus-induced monoclonal islet cell autoantibody.

Our previous finding that about 15% of newly diagnosed patients with Type 1 (insulin-dependent) diabetes mellitus had human cytomegalovirus genome in their lymphocytes and islet cell autoantibodies in their sera, suggests that autoimmune Type 1 diabetes is associated with persistent cytomegalovirus infection under certain circumstances. This investigation was initiated to see if cytomegalovirus can induce islet cell autoantibodies and if the autoantibodies react with any specific islet protein(s). Monoclonal antibodies were generated after immunizing Balb/c mice with human cytomegalovirus. When these monoclonal antibodies were tested for the presence of islet cell antibodies were tested for the presence of islet cell antibodies, one (MCMVA-51) of 13 monoclonal antibodies reacted strongly with the islets. The titer of islet cell antibodies was 1:2000. When this monoclonal antibody was reacted with the proteins from the solubilized fraction of human pancreatic islets using the western immunoblotting technique, a band with a molecular weight of 38 kilodalton was detected. The 38 kilodalton band was not observed when the monoclonal antibody was reacted with the proteins prepared from pancreatic islet tissues of rats and mice or from other human organs including stomach, liver, spleen and brain, indicating that the 38 kilodalton protein is human islet cell-specific. It is concluded that human cytomegalovirus can induce islet cell antibodies that react with a 38 kilodalton human islet cell protein and that this protein component may represent islet cell-specific target antigens associated with persistent cytomegalovirus infection.

Animals↗

PubMed1990

A cytotoxic monoclonal autoantibody from the BB rat which binds an islet cell surface protein.

The BB rat provides an excellent animal model for type 1 (insulin-dependent) diabetes mellitus. Cytotoxic autoantibodies against pancreatic beta cells have been found in the sera of both patients with type 1 (insulin-dependent) diabetes and BB rats. These antibodies have been implicated in the pathogenesis of the disease. In this study, a monoclonal autoantibody, designated KT1, has been developed by the fusion of spleen cells from a BB rat and a mouse myeloma cell line. KT1 was found to be of the immunoglobulin M isotype and reacted specifically with islet cells. In microcytotoxicity assays KT1 was shown to mediate complement-dependent lysis of approximately 30% of a rat insulinoma cell line and 25% of rat pancreatic islet cells in culture. It did not cause lysis of the other cell lines tested. KT1 has been demonstrated, by indirect immunofluorescence, to bind specifically to a cell surface antigen on live and acetone-fixed islet cell cultures from Wistar rat neonates and to rat insulinoma cells. Western blotting experiments revealed reaction to a 68-kDa protein from rat insulinoma cell extracts. This monoclonal antibody may have clinical relevance as it exhibits properties similar to the islet cell surface antibodies present in the sera of BB rats.

Animals↗

PubMed1990

Two amino acids, Phe 16 and Ala 776, on the polyprotein are most likely to be responsible for the diabetogenicity of encephalomyocarditis virus.

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to differ from the non-diabetogenic B variant (EMC-B) by 14 nucleotides out of 7829 bases. Similar approaches with a new nondiabetogenic variant, EMC-DV1, obtained by plaque purification of the EMC-D variant stock pool, enabled us to narrow down further the possible genomic area responsible for the diabetogenicity of EMC virus. EMC-DV1 does not induce interferon in vitro, differing from the highly interferon-inducing EMC-B. The complete nucleotide sequence of EMC-DV1 was determined by RNA-dependent DNA sequencing and cDNA sequencing. The genomic size and organization of EMC-DV1 are similar to those of EMC-D and EMC-B, with a long open reading frame encoding a polyprotein of 2292 amino acids. Comparative analyses of sequence information as well as biological activities of EMC-DV1 with EMC-D and EMC-B suggest that (i) the diabetogenicity is apparently distinct from the ability to induce interferon, which is probably due to the single U base insertion at position 765 in EMC-B, and (ii) the diabetogenicity of EMC virus is most probably controlled by one or both of two amino acids, Phe 16 (on the leader peptide) and Ala 776 (152nd amino acid on the VP1) on the polyprotein.

Alanine↗

PubMed1990

Role of macrophages in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from SJL/J mice infected with the D variant of encephalomyocarditis virus (EMC-D virus) showed lymphocytic infiltration with moderate to severe destruction of beta cells. Immunohistochemical staining of the islet sections with several monoclonal antibodies, anti-Mac-1, anti-Mac-2, and F4/80 for macrophages, anti-L3T4 for helper/inducer T cells, and anti-Lyt2 for cytotoxic/suppressor T cells revealed that the major population of infiltrating cells at the early stage of viral infection was Mac-2-positive macrophages. In contrast, macrophages detected by anti-Mac-1 and F4/80 monoclonal antibodies were not found at the early stage of viral infection but were found at intermediate and late stages of viral infection. Helper/inducer T cells and cytotoxic/suppressor T cells also infiltrated the islets at intermediate and late stages of viral infection. Short-term treatment of mice with silica prior to viral infection resulted in an enhancement of beta-cell destruction, leading to the development of diabetes. In contrast, long-term treatment of mice with silica resulted in complete prevention of diabetes caused by a low dose of viral infection and a significant decrease in the incidence of diabetes caused by an intermediate or high dose of viral infection. Furthermore, depletion of macrophages by a specific monoclonal antibody (anti-Mac-2) resulted in a much greater decrease in the incidence of diabetes caused by an intermediate dose of viral infection. However, suppression of helper/inducer T cells and cytotoxic/suppressor T cells, by anti-L3T4 and anti-Lyt2 antibodies, respectively, did not alter the incidence of diabetes. On the basis of these data, it is concluded that macrophages, particularly Mac-2-positive macrophages, play a crucial role in the process of pancreatic beta-cell destruction at the early stage of encephalomyocarditis D virus infection in SJL/J mice.

Animals↗

PubMed1990

Studies on autoimmunity for initiation of beta-cell destruction. VI. Macrophages essential for development of beta-cell-specific cytotoxic effectors and insulitis in NOD mice.

NOD mice were treated with silica (which is selectively toxic to macrophages) from 4 or 20.5 wk of age. Syngeneic neonatal pancreases were transplanted into the renal subcapsular space of the NOD mice at 21 wk of age. Silica treatment was continued until 24 wk of age, and then the mice were killed for examination of islet morphology. Neither the islets in transplanted pancreases nor the host pancreatic islets from the early long-term silica-treated animals revealed insulitis. In contrast, most of the islets in transplanted pancreases from the late short-term silica-treated animals showed severe insulitis and beta-cell necrosis, as did the host islets. A further experiment was performed to compare the effect of late short-term silica treatment with that of anti-L3T4-antibody treatment of the same time and duration. In contrast to the late short-term silica-treated animals, the transplanted pancreases in the anti-L3T4-antibody-treated animals revealed intact islets, although most of the host islets showed insulitis. The control group, which received no treatment but did receive neonatal pancreases, revealed severe insulitis and beta-cell necrosis of both transplanted and host islets. These results suggest that early macrophage depletion can abolish the development of beta-cell-specific immunologic effectors but that late macrophage depletion, after the development of insulitis, does not affect the destruction of beta-cells by preexisting effectors other than macrophages. We conclude that macrophages are essential for the development of beta-cell-specific cytotoxic effectors in the initial phase of insulitis in NOD mice.

Animals↗

PubMed1990

Studies on autoimmunity for initiation of beta-cell destruction. V. Decrease of macrophage-dependent T lymphocytes and natural killer cytotoxicity in silica-treated BB rats.

Administration of silica, which is selectively toxic to macrophages, to young BB rats resulted in the prevention of insulitis and diabetes. However, the mechanism leading to the prevention of an autoimmune process in silica-treated BB rats is not known. This study was undertaken to investigate the mechanism involved in prevention of insulitis and diabetes. Neonates of diabetes-prone BB (DPBB) rats injected with concanavalin A (ConA)-activated spleen cells from silica-treated DPBB (STDPBB) rats did not develop insulitis or diabetes, whereas DPBB neonates injected with ConA-activated spleen cells from the untreated DPBB rats developed both insulitis and diabetes. Not only was there a decrease of natural killer (NK) cell activity in splenocytes from STDPBB rats, but there was also a significant reduction in the number of immunocytes such as T lymphocytes (helper/inducer and cytotoxic/suppressor) and NK cells. The number of macrophages in both spleen and peripheral blood was significantly decreased in STDP rats compared with untreated DP rats. In contrast to the changes in T lymphocytes and NK cell activity, there was no change in target beta-cells in STDPBB rats with regard to the susceptibility to adoptive transfer of insulitis. It is concluded that the prevention of insulitis and diabetes in STDPBB rats is due to a decrease in macrophage-dependent T lymphocytes and NK cell cytotoxicity.

Animals↗

PubMed1989

Inherited diseases in North American Mennonites: focus on Old Colony (Chortitza) Mennonites.

The patterns of migration and the genetic disorders occurring among North American Mennonites are reviewed, and inherited conditions recently recognized in a religious and genetic isolate, the Old Colony (Chortitza) Mennonites, are described. Old Colony Mennonites are of Dutch/German origin and descend from approximately 400 founding families who settled in the Old Colony, Chortitza (the Ukraine, USSR) in the late 1700s, and then migrated to Canada and Central and South America in the past century. We investigated over 6 generations of a Canadian Old Colony kindred in which there was extensive intermarriage, and in whom 28 individuals developed diabetes mellitus. Insulin-dependent diabetes mellitus (IDDM) occurred in 14 affected individuals in 10 closely related sibships; the 11 living IDDM patients were all concordant for the immunogenetic marker HLA-DR4. Fourteen close relatives had other disorders of carbohydrate metabolism, including gestational diabetes and non-insulin-dependent diabetes mellitus. Other close relatives had autoimmune diseases, including rheumatoid arthritis, hyper- and hypothyroidism, multiple sclerosis, and red cell aplasia. Other inherited diseases, including Alport syndrome, congenital defects, and inborn errors of metabolism were also found in the kindred. In the almost exclusively (99%) Old Colony Mennonite public health district in which the kindred was ascertained, there were multiple cases of Tourette syndrome, of malformations (including congenital heart defects and cleft lip +/- palate), and familial clusters of inborn errors of metabolism. We report this Old Colony (Chortitza) Mennonite isolate because 1) there are large familial aggregations of tissue-specific autoimmune diseases, malformations, inborn errors of metabolism, and of some other conditions whose genetic basis is still unknown; 2) there are multiple cases of rare genetic conditions, 3) we have established a computerized genealogic data base on over 1,000 kindred members as well as a cryopreserved lymphocyte/DNA bank on over 100 closely related individuals with various genetic conditions; and 4) this religious isolate, which extends across North, Central, and South America, offers an excellent opportunity for studying the epidemiology and molecular genetics of both common and rare inherited diseases.

Adrenal Hyperplasia, Congenital↗

PubMed1989

Coxsackie B virus assays in IDDM families: the GAW5 data on antibody prevalence.

There is evidence that infection by Coxsackie viruses can serve as an environmental "trigger" for insulin-dependent diabetes mellitus (IDDM). This background information is summarized briefly, and the data in the IDDM component of Genetic Analysis Workshop 5 (GAW5) concerning serum antibodies to Coxsackie B viruses are described.

Antibodies, Viral↗

PubMed1989

Genomic differences between the diabetogenic and nondiabetogenic variants of encephalomyocarditis virus.

Plaque purification of the M variant of encephalomyocarditis (EMC-M) virus resulted in the isolation of two stable variants. One is a highly diabetogenic D variant (EMC-D) and the other is a nondiabetogenic B variant (EMC-B). The cDNA of EMC-D and EMC-B genomes were cloned and seven overlapping cDNA clones were selected to cover the entire genome except the 5'-end 310 bases which were determined by RNA-dependent DNA sequencing and enzymatic RNA sequencing. Each clone was restriction-mapped, subcloned, and sequenced. The genomes of EMC-D and EMC-B are composed of 7829 and 7825 bases, respectively. Both genomes contain a long open reading frame of 6876 nucleotides starting at position 830 on the consensus sequence, which encodes a polyprotein of 2292 amino acids. The sequences of EMC-D and EMC-B differ by two deletions, one insertion, and eight point mutations. The first deletion of 3 nucleotides is located in the 5' poly(C) tract where EMC-B has 127 nucleotides compared with 130 nucleotides in EMC-D. The second deletion in EMC-B involves 2 nucleotides at the 3'-end polyadenylation site. A single base insertion of U occurs at the 5' noncoding region of EMC-B. The eight point mutations are located in the polyprotein coding region. Two are silent and are each located in the structural gene 1B and in the nonstructural gene 2B. The remaining six mutations, one on the L gene and the other five on the 1D gene, introduce respective amino acid changes. It is concluded that the diabetogenic EMC-D viral genome (7829 bases) differs from the nondiabetogenic EMC-B viral genome (7825 bases) by 14 nucleotides out of 7829.

Amino Acid Sequence↗

PubMed1989

Identification and production of monoclonal antibodies against a discriminating protein molecule between B and D variants of encephalomyocarditis virus.

The protein profiles of two EMC virus variants B and D in the infected L929 fibroblast lysates were examined using one- and two-dimensional gel electrophoresis. Both variants yielded a protein molecule of similar molecular weight but differing in its isoelectric point (pI). The B variant lysate yielded a molecule with pI congruent to 7.5 whereas the same molecule from the D lysate focused at pI congruent to 5.2. A monoclonal antibody (MCA) produced against pI congruent to 5.2 fraction of the D variant successfully detected viral antigens in the D variant infected fibroblasts with only background cross reactivity with the B variant infected cells. This MCA also detected D viral antigen(s) in organ sections obtained from D but not from B variant infected mice. This study therefore suggests a clear shift in the pI of a 23 kD protein molecule serving as a possible discriminating antigen between the B and D variants of EMC virus.

Animals↗

PubMed1989

Viruses as a triggering factor of type 1 diabetes and genetic markers related to the susceptibility to the virus-associated diabetes.

Type 1 diabetes results from the destruction of insulin-producing pancreatic beta cells. Genetic and environmental factors are implicated in the beta cell destruction. As environmental factors affecting the induction of type 1 diabetes, diabetogenic viruses, chemicals, toxins, and diet are likely candidates as either primary injurious agents of beta cells or triggering agents for the induction of autoimmunity. Regarding viruses as a triggering factor of type 1 diabetes, there are at least two different pathogenic mechanisms in virus-induced diabetes: cytolytic infection of beta cells, leading to their destruction, and triggering of autoimmunity, leading to the autoimmune-mediated destruction of beta cells. Since there is no correlation between the induction of antibodies to Coxsackie B viruses and the presence of islet cell autoantibodies in patients with type 1 diabetes, the induction of diabetes by Coxsackie B viruses may be due to cytolytic infection of beta cells rather than an autoimmune response. In contrast, rubella virus and cytomegalovirus (CMV) do appear to be somehow associated with autoimmune type 1 diabetes since there is a strong correlation between the presence of islet cell autoantibodies and persistent infections. Regarding genetic factors, there are distinct markers related to the susceptibility to Coxsackie B4 virus-associated type 1 diabetes and CMV-associated type 1 diabetes. Four specific DNA restriction endonuclease fragments (BamHI-DQ-beta 6.6, TaqI-DR-beta 4.3, TaqI-DR-beta 2.5 and TaqI-DR-beta 1.5 kb) are related to the susceptibility to Coxsackie B4 virus-associated type 1 diabetes while six specific DNA restriction endonuclease fragments (BamHI-DQ-alpha 12.5, -beta 3.7 and -beta 3.2 kb, TaqI-DQ-alpha 7.2, -beta 7.2 and -beta 5.4 kb) are related to the susceptibility to CMV-associated type 1 diabetes.

Animals↗

PubMed1989

Scrapie-induced diabetes mellitus in hamsters.

Scrapie-infected hamsters had slightly elevated non-fasting plasma glucose levels, markedly abnormal glucose tolerance tests, and impaired release of insulin in response to a glucose load. Plasma cortisol levels were essentially the same in infected and uninfected animals. Histological examination of the pancreas revealed no morphological changes in infected animals with no alteration in distribution of cells secreting insulin, glucagon and somatostatin. In contrast, brains of scrapie-infected animals had the diffuse vacuolation typical of spongiform encephalopathy. These experiments suggest that scrapie-induced diabetes mellitus in hamsters may result from damage to the central nervous system.

Animals↗

PubMed1989

Molecular identification of diabetogenic viral gene.

The best evidence that viruses have a causative role in the pathogenesis of insulin-dependent diabetes mellitus comes from experiments in mice infected with encephalomyocarditis (EMC) virus. When SJL/J male mice were inoculated with a highly diabetogenic EMC-D virus, diabetes developed in 95% of the animals. In contrast, none of the mice inoculated with a nondiabetogenic EMC-B virus became diabetic. Tissue culture experiments showed that EMC-B induces considerable amounts of interferon, whereas EMC-D does not. Despite these differences, EMC-D and EMC-B could not be distinguished antigenically by a sensitive plaque-neutralization assay. Furthermore, the buoyant density in CsCl density gradients and the capsid proteins of these two variants on polyacrylamide gels could not be distinguished. Molecular-hybridization studies with radiolabeled DNA complementary to EMC-D and EMC-B RNAs failed to distinguish them. Determination of complete nucleotide sequences of EMC-D and EMC-B revealed that EMC-D (7829 bases) differs from EMC-B (7825 bases) by only 14 nucleotides. The differences consist of two deletions of five nucleotides, one base insertion, and eight point mutations. The first deletion of three nucleotides and the second deletion of two nucleotides are located in the 5'-poly(C) tract and the 3'-end polyadenylation site, respectively. One base insertion in EMC-B occurs in the 5'-noncoding region. The eight point mutations are located in the polyprotein-coding region. Two of them are silent, whereas the other six mutations, one located on the L gene and five on the VP1 gene, introduce amino acid changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation.

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.

Biotechnology↗