Molecular biology & medicine最新文献

Autologous implants of genetically modified fibroblasts may be an ideal approach to gene replacement therapy. However, primary fibroblasts have limited life span and their use for gene therapy is questionable. We have developed a rat model to evaluate the feasibility of this approach. Primary fibroblasts initiated from rat skin biopsies were transfected with plasmid DNA. The transkaryotic fibroblasts were implanted into the original rat donors. A plasmid-encoded gene product, human growth hormone, was detected in the donor rats' circulation but less than 1% of the expected amount was recovered. Implantation at intraperitoneal, intramuscular or subcutaneous sites were effective in delivering the novel gene product. Fluctuating levels of human growth hormone were detected for over 6 months but their determination was rendered unreliable due to interference by extremely high titers of antibodies developed against the human growth hormone. In some cases, the antibody titer continued rising for more than 6 months, indicative of the continued presence of the antigen. One of the recipients developed two intra-abdominal fibrosarcomas that were shown to be derived from the implanted cells. In conclusion, this model demonstrates that autologous fibroblast implantation is a feasible approach to delivering novel gene products lasting for several months. Strong and prolonged immuno-reaction against the gene product will occur if the recipient's immune system is naive to the replacement product, as would occur in patients with CRM- mutations. The potential for neoplastic development from the implanted fibroblasts should prompt further evaluation of the long-term safety in gene replacement therapy.

Several mutations in the human lipoprotein lipase (LPL) gene have been shown to underlie LPL deficiency. These mutations occur in patients who are mainly of European descent, and comprise a single base transition causing a premature stop codon, four separate amino acid substitutions and two large gene rearrangements. Together they account for approximately 40% of the LPL alleles in a cohort of 50 patients whose DNA has been examined in this laboratory. We now report on a new mutation in exon 3 of the LPL gene from a South African subject of South-east Asian extraction. This mutation comprises a six base-pair insertion at the site of a single base deletion. The net insertion of five base-pairs at amino acid positions 102 to 103 causes a shift in the reading frame, generating 44 amino acid residues of random sequence and a premature stop codon within exon 4. This mutation is predicted to result in the synthesis of a markedly truncated protein and is the cause of the enzyme deficiency in our patient.

The centromeres and short arms of the human acrocentric chromosomes have in common several families of tandemly repeated DNA. Recent analyses have revealed that, within some of these families, clearly distinct subfamilies have evolved that are unique to one or a subset of the different acrocentric chromosomes. The existence and maintenance of subfamilies common to different chromosomes entail a process of regular exchange between the non-homologous chromosomes. This process is suggested for the evolution of an alpha satellite subfamily recently found on chromosomes 13, 14 and 21. The presence of this alpha subfamily may allow pairing between these chromosomes leading to the observed non-random participation of these chromosomes in t(13q14q) and t(14q21q) Robertsonian translocations. The available data also indicate a very similar molecular organisation of the cen-pter region for chromosomes 13 and 21. This latter feature may further allow the two chromosomes to undergo a relatively extensive degree of meiotic pairing (in a manner analogous to that seen in the pseudoautosomal regions of the X and Y chromosomes), thus predisposing these two chromosomes to errors in meiotic segregation and non-disjunction.

All nucleated animal cells synthesize heme to provide the prosthetic group of respiratory cytochromes. Large amounts of heme are synthesized by erythroid cells for hemoglobin production and by liver cells for drug-induced cytochromes P450. This review focuses on the first enzyme of the heme biosynthetic pathway, 5-aminolevulinate synthase (ALAS), which catalyzes the rate-controlling step in liver and possibly other tissues. We report that there are two distinct human genes for ALAS: one, a housekeeping gene, is probably ubiquitously expressed while the other is active only in erythroid tissue. By contrast it has been reported that, for porphobilinogen deaminase, the third enzyme of the heme pathway, there is a single human gene with two promoters; one functional in all tissues, the other erythroid specific. In liver, transcription of the housekeeping ALAS gene is induced by drugs and repressed by heme. Heme also acts in a novel way to prevent transport of ALAS into mitochondria, its site of function. Porphyrias result from inherited defects in enzymes of the heme pathway subsequent to ALAS and the molecular abnormality is now known for the most common subtype of acute intermittent porphyria. In developing red cells, levels of ALAS are regulated by increased gene transcription and by a post-transcriptional mechanism, in which iron most probably controls translation of erythroid ALAS mRNA through an iron-responsive element identified in the 5' untranslated region of the mRNA. The human erythroid ALAS gene is located on the X-chromosome, suggesting that a defect in this gene may be responsible for X-linked sideroblastic anemias.

We have used a moderate repeat probe and a number of single copy DNA probes of varying sizes to compare different approaches to non-radioactive in situ hybridization. We have compared the ease and speed of the methods, the sensitivity, resolution, reproducibility, the availability and costs of reagents, and the potential for clinical application. Following biotinylation or mercuration, the probes were hybridized to human metaphases and nuclei and detected by different affinity systems. Visualization of signals was by brightfield, phase contrast, fluorescence or reflection contrast microscopy. As a result of our study, we recommend two simple and reliable methods using the biotinylation approach with either the avidin-peroxidase conjugate and diaminobenzidine detection and reflection contrast microscopy, or the streptavidin-alkaline phosphatase conjugate and bromochlorodinolyl phosphate nitroblue tetrazolium chloride detection using phase contrast microscopy.

The ability to diagnose human diseases at the DNA level has become possible because of a rapid development in DNA technology, particularly in the area of detection of single base substitutions. Mutations in the genomic DNA of a particular gene may be inferred indirectly using linkage analysis and restriction fragment length polymorphisms. However, direct detection of the mutation is the more favourable approach. The advent of the polymerase chain reaction to amplify specific regions of genomic DNA or mRNA has enhanced the speed and sensitivity of many of the screening and diagnostic procedures. Screening methods have been developed that will detect at least 70% and, with some methods, close to 100% of all mutations. The methods include ribonuclease A cleavage, denaturing gradient gel electrophoresis, chemical cleavage of mismatch and direct sequencing. Choice of method is based on a number of factors and will depend on the structure of the gene to be analysed. Following identification of a mutation using one of the screening procedures, prenatal diagnosis and carrier testing can be offered. The overall aim is to develop a method that has the potential to determine the mutation present in an index case of a previously untested family in a few days, thus allowing any other relevant family member to be tested.

Exposure of any living cell to a change in environmental conditions such as increased growth temperature results in the induction of stress, or "heat shock", proteins. Proteins identified in such experiments have subsequently been shown to be ubiquitous components of all cells and to perform essential functions during normal cell growth in addition to their role during stress. It has recently been recognized that members of stress protein families play an important role in the immune response to a wide variety of infections, and the highly conserved nature of such proteins has led to the suggestion that they may also be immune targets in autoreactive responses. In this article we review the role of stress proteins in the immune response to infection and discuss the evidence which suggests that lymphocytes recognizing conserved determinants on such antigens may be associated with autoimmune diseases.

We report a revised sequence of a cDNA that encodes a human galactose-1-phosphate uridyl transferase. The cDNA is 1295 bases in length and encodes a 43,000 Mr protein. The sequence was derived from a cDNA clone isolated from a transformed human lymphoblast cell line and amplified in a polymerase chain reaction. The revised sequence reveals a higher degree of amino acid conservation between the human enzyme and the homologous enzymes from Escherichia coli and yeast than was previously thought to exist.

Cloning of the cystic fibrosis gene and the identification of the predominant disease-causing mutation did not only help in the understanding of this frequent disease, but was immediately followed by applications in direct gene diagnosis. We describe a method for the detection of the so-called delta F508 deletion, which accounts for 70% of the mutations: a polymerase chain reaction with two different combinations of oligonucleotide primers, which discriminate between mutant and wild-type alleles. This allele-specific amplification provides a rapid, non-radioactive and very reliable method for direct genotyping. Establishment of the procedure and its application in diagnosis are described. We further report preliminary data on the frequency of this mutation in German patients and its association with restriction fragment length polymorphism haplotypes.