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.     

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Ammonia formation in isolated rat liver mitochondria

Studies of isolated rat liver mitochondria were undertaken in order to evaluate the importance of glutamate transport, oxidation reduction state, and product inhibition on the rates of formation of ammonia from glutamate. Uptake and efflux of glutamate across the mitochondrial membrane were measured isotopically in the presence of rotenone. Efflux was stimulated by H+ in the mitochondrial matrix and was found to be first order with respect to matrix glutamate except when the matrix pH was unphysiologically low. The data suggest that the Km of matrix glutamate for efflux is decreased by H+. Matrix H+ also appeared to stimulate glutamate uptake, but the effect was to increase both the Km of medium glutamates and Vmax. Mitochondria were incubated at 15 and 28 degrees C with glutamate and malonate. Under these conditions, glutamate was metabolized only by the deamination pathway. Flux was evaluated by assay of ammonia formation. Oxidation reduction state was varied with ADP and uncoupling agents. Matrix alpha-ketoglutarate was varied either by the omission of malonate from the incubation media or by adding alpha-ketoglutarate to the external media. Influx and efflux of glutamate could be calculated from previously determined transport parameters. The difference between calculated influx and efflux was found to be equal to ammonia formation under all conditions. It was, therefore, possible to evaluate the relative contributions of oxidation reduction state, transport, and product inhibition as effectors of ammonia formation. The contribution of transport was relatively small while oxidation reduction state exerted a large influence. alpha-Ketoglutarate was found to be a potent competitive inhibitor of ammonia production and glutamate dehydrogenase. Inhibition of glutamate dehydrogenase by alpha-ketoglutarate was judged to be a potentially important modulator of metabolic fluxes.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.     

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.     

Expression of SV-40 T antigen in the small intestinal epithelium of transgenic mice results in proliferative changes in the crypt and reentry of villus-associated enterocytes into the cell cycle but has no apparent effect on cellular differentiation programs and does not cause neoplastic transformation

The mouse intestinal epithelium represents a unique mammalian system for examining the relationship between cell division, commitment, and differentiation. Proliferation and differentiation are rapid, perpetual, and spatially well-organized processes that occur along the crypt-to-villus axis and involve clearly defined cell lineages derived from a common multipotent stem cell located near the base of each crypt. Nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene were used to establish three pedigrees of transgenic mice that expressed SV-40 large T antigen (TAg) in epithelial cells situated in the uppermost portion of small intestinal crypts and in already committed, differentiating enterocytes as they exited these crypts and migrated up the villus. T antigen production was associated with increases in crypt cell proliferation but had no apparent effect on commitment to differentiate along enterocytic, enteroendocrine, or Paneth cell lineages. Single- and multilabel-immunocytochemical studies plus RNA blot hybridization analyses suggested that the differentiation programs of these lineages were similar in transgenic mice and their normal littermates. This included enterocytes which, based on the pattern of [3H]thymidine and 5-bromo-2'-deoxyuridine labeling and proliferating nuclear antigen expression, had reentered the cell cycle during their migration up the villus. The state of cellular differentiation and/or TAg production appeared to affect the nature of the cell cycle; analysis of the ratio of S-phase to M-phase cells (collected by metaphase arrest with vincristine) and of the intensities of labeling of nuclei by [3H]thymidine indicated that the duration of S phase was longer in differentiating, villus-associated enterocytes than in the less well-differentiated crypt epithelial cell population and that there may be a block at the G2/M boundary. Sustained increases in crypt and villus epithelial cell proliferation over a 9-mo period were not associated with the development of gut neoplasms--suggesting that tumorigenesis in the intestine may require that the initiated cell have many of the properties of the gut stem cell including functional anchorage.