Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.     

Characterization of calcium-activated, phospholipid-dependent protein kinase from rat serosal mast cells and RBL-1 cells

Evidence is presented that rat serosal mast cells and cells of the rat basophilic leukemia line, RBL-1, each contain a calcium-activated, phospholipid-dependent protein kinase. The enzymes are very similar in their activation requirements to the calcium-dependent enzymes termed protein kinase Cs in brain. The enzyme is selectively stimulated by diolein and phosphatidylserine and is inhibited by several local anesthetics. The Ka for Ca2+ is 1.0 X 10(-3) M and 1.5 X 10(-4) M in mast cells and RBL-1 cells, respectively. The enzyme in mast cells is rapidly activated and apparently changed in its intracellular distribution when intact mast cells are stimulated with 48/80, A-23187, and anti-IgE and 12-O-tetradecanoyl-13-acetate in combination.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.