Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.     

Comparison of non-mydriatic retinal photography with ophthalmoscopy in 2159 patients: mobile retinal camera study.

OBJECTIVE--To determine whether non-mydriatic Polaroid retinal photography was comparable to ophthalmoscopy with mydriasis in routine clinic screening for early, treatable diabetic retinopathy. DESIGN--Prospective study of ophthalmoscopic findings according to retinal camera screening and ophthalmoscopy and outcome of referral to ophthalmologist. SETTING--Outpatient diabetic clinics of three teaching hospitals and three district general hospitals. PATIENTS--2159 Adults selected randomly from the diabetic clinics, excluding only those registered as blind or those in wheelchairs and unable to enter the screening vehicle. MAIN OUTCOME MEASURES--Numbers of patients and eyes correctly identified by each technique as requiring referral with potentially treatable retinopathy (new vessel formation and maculopathy) and congruence in numbers of microaneurysms, haemorrhages, and exudates reported. RESULTS--Camera screening missed two cases of new vessel formation and did not identify a further 12 but indicated a need for referral. Ophthalmoscopy missed five cases of new vessel formation and indicated a need for referral in another four for other reasons. Maculopathy was reported in 147 eyes with camera screening alone and 95 eyes by ophthalmoscopy only (chi 2 = 11.2; p less than 0.001), in 66 and 29 of which respectively maculopathy was subsequently confirmed. Overall, 38 eyes received laser treatment for maculopathy after detection by camera screening compared with 17 after ophthalmoscopic detection (chi 2 = 8.0; p less than 0.01). Camera screening underestimated numbers of microaneurysms (chi 2 = 12.9; p less than 0.001) and haemorrhages (chi 2 = 7.4; p less than 0.01) and ophthalmoscopy underestimated hard exudates (chi 2 = 48.2; p less than 0.001). CONCLUSIONS--Non-mydriatic Polaroid retinal photography is at least as good as ophthalmoscopy with mydriasis in routine diabetic clinics in identifying new vessel formation and absence of retinopathy and is significantly better in detecting exudative maculopathy.     

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.     

Synthesis and degradation of collagens in skin of healthy and protein-malnourished rats in vivo, studied by 18O2 labelling.

To explore the effects of growth retardation, caused by restricted protein intake, on collagen turnover in the whole skin, Sprague-Dawley rats (n = 20) were labelled with 18O2 and fed on either an adequate (18%) or a low (3%) lactalbumin diet. Skin biopsies were obtained at intervals during the following 6 months. Independent groups of animals (n = 186) were used to determine the size of the 0.5 M-acetic acid-soluble and -insoluble collagen pools in the entire skin of healthy and malnourished rats. Collagen was estimated by measurement of hydroxyproline. Soluble-collagen synthesis rates were equivalent to 99 +/- 8 mumol of hydroxyproline/day in healthy animals and 11 +/- 2 mumol/day in malnourished rats. Insoluble-collagen synthesis rates were 32 and 5 mumol of hydroxyproline/day in the healthy and protein-depleted rats respectively. The degradation of soluble collagen amounted to 37 +/- 8 and 6 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Efflux of collagen from the soluble collagen, defined as the sum of the rate of soluble collagen that is degraded plus that which matures into insoluble collagen, was 70 +/- 8 and 11 +/- 2 mumol of hydroxyproline/day in the healthy and malnourished groups respectively. Insoluble collagen was not degraded in either group. The fraction of soluble collagen leaving the pool that was converted into insoluble collagen was 0.46 in both diet groups. It is concluded that the turnover of soluble collagen is markedly decreased with malnutrition, but degradation and conversion into insoluble collagen account for the same proportions of efflux from the soluble-collagen pool as in rapidly growing rats.     

Species-specific TNF induction of thymocyte proliferation

Summary Recombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1000 units/ml TNF were near optimal while concentrations 1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1 or . The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.Supported in part by Asahi Chemical Industry Co., Inc. and by USPHS Grants CA-24538, CA-15142 and CA-09072 awarded by the National Cancer Institute, Department of Health and Human Services