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101.
Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction 总被引:83,自引:0,他引:83
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. 相似文献
102.
Christopher J. Molloy Jeffrey D. Laskin 《Differentiation; research in biological diversity》1988,37(2):86-97
Adult mouse epidermis contains up to 11 distinct keratin polypeptides, as resolved by two-dimensional gel electrophoresis. These include both basic (Type II; 67-, 65-, 63-, 62-, and 60-kDa) and acidic (Type I; 61- to 59-, 54-, 52-, 49-, and 48-kDa) keratins that exhibit multiple isoelectric forms. Several, but not all, of these keratins, identified by immunoblotting, were found to be actively synthesized in the skin when assayed in short-term pulse-labeling experiments. When compared to the adult, newborn mouse epidermis expresses fewer keratin subunits. However, greater amounts of keratins associated with differentiated suprabasal cells and stratum corneum, which is more pronounced morphologically in the newborn, were identified. We also observed strain-specific differences in the expression of a Type I acidic keratin. This 61-kDa (pI, approx. 5.3) keratin was produced exclusively by the CF-1 mouse and, based on peptide mapping, appeared to be related to the acidic 59-kDa keratin that was identified in this strain as well as all other mouse strains. The 61-kDa keratin was not expressed in vitamin A-deficient animals, suggesting that its appearance may be related to a retinoid-dependent posttranslational modification. In comparison to keratin expression in vivo, primary mouse keratinocyte monolayer cultures maintained in low Ca2+ (less than 0.08 mM) did not express the terminal differentiation keratins of 67-kDa (basic) or 59-kDa (acidic), although enhanced synthesis of the 60-kDa (basic) and the 52-kDa and 59-kDa (acidic) keratins associated with proliferation were observed. In addition, a subpopulation of nonadherent cells was continuously produced by the primary keratinocyte cultures that expressed the 67-kDa (basic) keratin specific for terminal differentiation. When the keratinocyte cultures were induced to terminally differentiate with Ca2+, the overall pattern of keratin expression was not changed significantly. Taken together, these results provide further evidence for the variable nature of keratin expression in mouse epidermal keratinocytes under different growth conditions. 相似文献
103.
104.
Andreas Kiener William H. Orme-Johnson Christopher T. Walsh 《Archives of microbiology》1988,150(3):249-253
Intracellular levels of F390 (AMP and GMP adducts of the 5-deazaflavin cofactor F420) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F390 was detected in growing cells, hydrogen starved cells and CO2 starved cells prior to O2 contamination. Also, no F390 was found in hydrogen depleted cells after O2 treatment. Exposure of exponentially growing cells and CO2 starved cells to oxygen lead to the formation of F390 species; the increase in the detected amount of F390 was coupled to a decrease of the F420 level. As soon as anaerobiosis was reestablished F390 cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O2 exposure. After normal growth conditions were restored the level of detected methanopterin decreased again. 相似文献
105.
M. Jane Ehrke Richard L. X. Ho Kazuyoshi Hori 《Cancer immunology, immunotherapy : CII》1988,27(2):103-108
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 相似文献
106.
Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions 总被引:2,自引:2,他引:0
Leif Andersson Anne Lundén Sunna Sigurdardottir Christopher J. Davies Lars Rask 《Immunogenetics》1988,27(4):273-280
Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO
, DQ
, DQ
, DR
and DR
genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ
, DY
, and DY
, are reported. DZ
was assumed to correspond to the human DZ
gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ
, DQ
, DR
and DR
loci and the other one is composed of the DO
DY
, DY
, and TCPIB loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 ± 0.07. The fairly high recombination frequency observed between class 11 genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational hot spot in the bovine class II region. 相似文献
107.
108.
Summary A simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 . Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.Nomenclature as
specific area of a support (cm)
- Bi
Biot number
-
dimensionless
- Cb
oxygen concentration in the bulk liquid (mM)
-
C
b
C
b
*
-Ccr (mM)
- C
b
*
bulk oxygen concentration in equilibrium with air (mM)
- Ccr
critical oxygen concentration (mM)
- Cs
oxygen concentration in the solid phase (mM)
- dp
diameter or thickness of a support (cm)
- Deff
effective diffusivity of oxygen in the solid phase (cm2/s)
- km
membrane permeability of oxygen (cm/s)
- k
m
*
Deff/m
- kLaL
liquid phase mass transfer rate coefficient (1/s)
- ksas
solid phase mass transfer rate coefficient (1/s)
- (OUR)v
volumetric oxygen uptake rate (mmol O2/l)
- p
geometry parameter, p=0 for slab, p=1 for cylinder, p=2 for sphere
- Pd
oxygen penetration depth (cm)
-
P
d
oxygen penetration depth in the absence of external diffusion limitation (cm)
- Q
volumetric oxygen uptake rate,
(mmol O2/l·h)
-
specific oxygen uptake rate (mmol O2gm biomass (dry)·h)
- r
length coordinate (cm)
- rc
oxygen penetration depth for sphere (cm)
-
r
c
rc in the absence of external diffusion limitation (cm)
- r
c
*
oxygen penetration depth for cylinder (cm)
-
r
c
*
r
c
*
in the absence of external diffusion limitation (cm)
- rcom
combined mass transfer rate resistance (s)
- rd
location where Cs becomes zero or Ccr (cm)
- ri
radius of cylinder or sphere, half thickness of slab (cm)
- Usg
superficial gas velocity (cm/s)
- X
cell concentration (g/l)
Greek letters
Thiele modulus, dimensionless
- L, s
liquid and solid phase volume fraction, respectively, dimensionless
-
effectiveness factor
On sabbatical leave from KAIST, Seoul, Korea 相似文献
109.
Aromatic l-Amino Acid Decarboxylase Activity of the Rat Retina Is Modulated In Vivo by Environmental Light Maria Hadjiconstantinou 总被引:3,自引:1,他引:2
Zvani Rossetti Christopher Silvia Dimitrij Krajnc Norton H. Neff 《Journal of neurochemistry》1988,51(5):1560-1564
Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina is low in animals placed in the dark. When the room lights are turned on, activity rises for almost 3 h and reaches values that are about twice the values found in the dark. A study of the kinetics of the enzyme revealed that the apparent Km values for L-3,4-dihydroxyphenylalanine and pyridoxal-5'-phosphate were unchanged in light- and dark-exposed animals, whereas the Vmax increased in the light. Treating the animals with cycloheximide before exposure to light prevented the increase of enzyme activity. Immunotitration with antibodies to AAAD suggested that more enzyme molecules are present in the light than in the dark. When the room lights are turned off AAAD activity drops rapidly at first and then more slowly, suggesting that at least two processes are responsible for the fall of enzyme activity. Exposure to short periods of dark followed by light results in a rapid increase of AAAD activity. Mixing homogenates from light- and dark-exposed rats results in activity values that are less than expected, suggesting the presence of an endogenous inhibitor(s). These studies demonstrate that AAAD activity is modulated in vivo. 相似文献
110.
The effects of subacute treatment with cocaine on activities of cocaine N-demethylase, UDP-glucuronyltransferase (GT) toward 4-nitrophenol and phenolphthalein and sulfotransferase (ST) toward androsterone and 4-nitrophenol in livers from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. Hepatic metabolism of cocaine was different between the sexes (with males having higher N-demethylase activity) and the strains (with WKY rats having higher activity). The effects of subacute cocaine administration on the activity of cocaine N-demethylase were also sex- and strain-related. Whereas cocaine administration increased activity of hepatic N-demethylase in both female strains, it decreased activity in male WKY and had no effect on activity in male SHR. Sex and strain-related as well as cocaine-induced differences were also found in activities of hepatic GT toward 4-nitrophenol and phenolphthalein as well as in activity of hepatic ST towards andersterone and 4-nitrophenol. These results suggest that some of the individual variation in the effects of cocaine may be due to sex and genetic differences in the hepatic metabolism of cocaine and/or in sexually and/or/genetically-determined differences in how cocaine affects hepatic metabolism of other xenobiotics. 相似文献