Duplications and deletions of Vh genes in inbred strains of mice

evolution of variable region (Vh) gene family copy number and polymorphism was investigated by the analysis of the immunoglobulin heavy chain variable region (Igh-V) locus in 74 inbred strains and substrains of mice. Several strains were found to have slight differences from Igh-V haplotypes previously identified, usually of a single Vh gene family. These results indicate that the evolution of copy number in the mouse Igh-V locus proceeds largely by the accumulation of incremental changes, reflecting the clustered organization of the mouse Igh-V locus. We have found no evidence of very large or frequent duplication or deletion events indicative of rapid expansion or contraction processes. The existence of one or more particularly large Vh gene families most likely reflects random copy number variation, rather than selection for the amplification of their members. The identification of strains with recombinant Vh gene arrays demonstrates that recombination, both within and between haplotypes, appears to be the predominant mechanism generating the high restriction fragment length polymorphism in the Igh-V locus.Abbreviations used in this paper Igh-V immunoglobulin heavy chain variable region locus - Vh heavy chain variable region gene - Dh heavy chain diversity region gene - V immunoglobulin kappa light chain variable region gene - V T-cell receptor beta chain variable region gene     

Optimization and control of a continuous stirred tank fermenter using learning system

A variable structure learning automaton is used as an optimization and control of a continuous stirred tank fermenter. The algorithm requires no modelling of the process. The use of appropriate learning rules enables to locate the optimum dilution rate in order to maximize an objective cost function. It is shown that a hierarchical structure of automata can adapt to environmental changes and can also modify efficiently the domain of variation of the control variable in order to encompass the optimum value.List of Symbols f Random number - F Dimensionless flow rate (F/V ₀) - F m³/h Flow rate - F ₀ m³/h Inlet flow rate - J Objective function - K _i Dimensionless constant in Eq. (3) (k _i/s₀) - k _i · kg/m³ Substrate inhibition constant in Haldane model - K _m Dimensionless constant in equation (3) (k _s/s₀) - k _m kg/m³ Substrate inhibition constant in Haldane model - L Number of levels of the hierarchical system of automata - N Number of possible control actions - p Probability - S Dimensionless substrate concentration (s/s ₀) - s kg/m³ Substrate concentration - T Dimensionless sampling period - t h Time - v Dimensionless volume (V/V ₀) - V m³ Liquid volume in fermenter - W Input to the stochastic automaton - X Dimensionless biomass concentration - x kg/m³ Biomass concentration - Y Biomass/substrate yield coefficient - Weighting factor in Eq. (4) - Dimensionless specific growth rate (/ ^*) - ^* h^–1 Maximum specific growth rate - h^–1 Specific growth rate - Dimensionless time ( t)     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Genetic chasing of T helper cell idiotype and allotype genes

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1⁺, 2.3^–B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1⁺, 2.3^–B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1 ^b genes (B6) and not with Igh-1 ^b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1⁺, 2.3^–B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1 ^b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1⁺, 2.3^–B6 T cells are produced by genes also linked to Igh-1 ^b -allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.Abbreviations used in this paper BCGF B cell growth factor - B6 C57B1/6 - C_H constant region of immunoglobulin heavy chain - Con A concanavalin A - FCS fetal calf serum - Id idiotype - Ig immunoglobulin - LPS lipopolysaccharide - M mouse - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MRBC mouse red blood cells - NMS normal mouse serum - NP nitrophenacetyl - NRS normal rabbit serum - PFC plaque forming cell - R rabbit - Tcf T cell factor - Tcr T cell receptor - TNP Trinitrophenol - V_H variable region of Ig heavy chain Definitions of terms used in this paper: T-cell idiotypes, structures on T-cell membranes or released T-cell molecules detected by an anti-idiotypic antiserum (5936) produced against specific immunoglobulin idiotypes. The 5936 T-cell idiotypes are related to the specific binding of IA^k gene products by certain Igh-1^b T cells. T cell allotypes, structures on T-cell membranes or released T-cell molecules detected by an antiserum (6036) produced against 5936 idiotype-bearing T-cell molecules. The 6036 T-cell allotypes are related to the binding by Igh-1^b T cells of all Ia gene products tested, and they are non-cross-reactive with immunoglobulin allotypes.     

Recombination between kappa chain genetic markers and the Lyt-3 locus

Recombination has been detected for the first time between chromosome 6 loci controlling kappa chain expression in normal mouse serum immunoglobulin and the Lyt-3 locus. The recombination event occurred at the 26th or 27th backcross generation during the derivation of the Lyt-2a, Lyt-3a-congenic line B6.PL(85NS). The line is now homozygous for the Lyt-2a, Lyt-3a allele(s) at N30F13 and homozygous animals express the Igk-Ef1b allele derived from C57BL/6. The frequency of recombination has been estimated to be 0.30% based on the present results and previous studies in which no recombination was detected. The results rule out the hypothesis that the Lyt-3 locus itself controls the light chain phenotype observed in normal serum immunoglobulin.     

BALB/c antiarsonate idiotypes: Gene complementation necessary for expression

The expression of two idiotype (id) families (5AF6 and 3C6) associated with the BALB/c p-azophenylarsonate-specific antibody response was examined in 11 mouse strains. Eight strains produced some of one or the other of these two id families with the mean percent expression in the anti-Ar responses of id(+) strains ranging from 8 to 43% for the 5AF6 and from 2 to 10% for the 3C6 idiotype. Four strains of mice (C58, AKR, PL, and RF) thought to have Lyt-3.1-linked V_L repertoire differences from other mouse strains (Lyt-3.2) were tested for their capacity to contribute to 5AF6 and 3C6 id expression. The RE strain was capable of producing 5AF6 id and small amounts of 3C6 id. Tests of Lyt-3.1 congenic strains C.AKR (AKR Lyt-3.1 on a BALB/c background) and C.C58 (C58 Lyt-3.1 on a BALB/c background) showed that C.AKR could produce 5AF6 id while C.C58 could not. 3C6 id expression was present but depressed in C.C58 mice compared with the high 3C6 id expression in C.AKR. Breeding studies mating C.C58 (bearing the required Igh-C^a-linked V _H genes) to other 5AF6(–) strains showed that gene complementation could result in 5AF6 expression in F₁ offspring. 5AF6(–) strains capable of complementation included CBA/J, C57BL/6J, AKR/J, and PL/J. C58/J (from which C.C58 were derived) was the only tested strain that failed to complement for 5AF6 id expression. Additional matings between C58/J[5AF6(–)] and CBA/J[5AF6 (–)] showed F1 offspring could produce 5AF6 id, indicating that C58/J can contribute functional V _H genes necessary for 5AF6 id expression. Depressed expression of 5AF6 and 3C6 id was noted in mice where the C58/J-derived Lyt-3.1 genotype was present. The possibility that the depression of 5AF6 and 3C6 id expression derived from C58/J mice was due to regulatory influences rather than a lack of the V _L structural genes is discussed.Abbreviations used in this paper Ar p-azophenylarsonate - DNP dinitrophenol - H heavy - HIS hyperimmune suppressed - id idiotype - Ig immunoglobulin - IEF isoelectric focusing - k kappa - L light - PC phosphorylcholine - S.D. standard deviation     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

The gene encoding human transmembrane secretory component (locus PIGR) is linked to D1S58 on chromosome 1

The human transmembrane secretory component (SC or poly-Ig receptor, PIGR) is expressed basolaterally on glandular epithelial cells and is responsible for the external translocation of polymeric IgA and IgM. SC is hence a key molecule in antibody protection of mucosal surfaces. The human SC gene (locus PIGR) is located on chromosome 1 (1q31–q41). Here we present the first genetic linkage study of PIGR versus syntenic markers, including D1S58 and F13B, which have been previously regionalized to 1q31–q32 and 1q31–q32.1, respectively. We found that PIGR is closely linked to D1S58 (lods + 5.06 at _max = 0.06, without sex difference). PIGR versus F13B showed + 1.46 at _max = 0.25 for both sexes combined. A recombination of 0.06 between F13B and D1S58 (lods + 2.24) was in contrast to a previously published study giving _max = 0.22 (lods + 3.9), the combined lods being 5.6 at _max = 0.20. The progeny of a triply heterozygotic female indicated that PIGR is the flanking locus, therefore suggesting a cen-F13B-D1S58-PIGR-qter gene sequence on human chromosome 1. Only negative lod scores to RH, C8@, and PGM1 on 1p, and FY on proximal 1q, were found. Current combined Norwegian allele frequencies were estimated for PIGR to be A1 = 0.63, A2 = 0.37 (370 chromosomes), and for D1S58 to be A1 = 0.44, A2 = 0.56 (218 chromosomes).     

Rapid cloning of any rearranged mouse immunoglobulin variable genes

Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologist. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5 and 3 universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36–60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny.The nucloetide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number U32111     

Enzyme production in a cell recycle fermentation system evaluated by computer simulations

Enzyme production in a cell recycle fermentation system was studied by computer simulations, using a mathematical model of -amylase production by Bacillus amyloliquefaciens. The model was modified so as to enable simulation of enzyme production by hypothetical organisms having different production kinetics at different fermentation conditions important for growth and production. The simulations were designed as a two-level factorial assay, the factor studied being fermentation with or without cell recycling, repression of product synthesis by glucose, kinetic production constants, product degradation by a protease, mode of fermentation, and starch versus glucose as the substrate carbon source.The main factor of importance for ensuring high enzyme production was cell recycling. Product formation kinetics related to the stationary growth phase combined with continuous fermentation with cell recycling also had a positive impact. The effect was greatest when two or more of these three factors were present in combinations, none of them alone guaranteeing a good result. Product degradation by a protease decreased the amount of product obtained; however, when combined with cell recycling, the protease effect was overshadowed by the increased production. Simulation of this type should prove a useful tool for analyzing troublesome fermentations and for identifying production organisms for further study in integrated fermentation systems.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - c starch concentration (g/l) - c ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - e intrinsic intracellular amylase concentration (g product/g cell mass) - E extracellular amylase concentration (g/l) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA/cell - K ₁ intracellular repression constant - K ₂ intracellular repression constant - K _s Monod saturation constant (g/l) - k ₃ product excretion rate constant (h^–1) - k _I translation constant (g product/g mRNA/h) - k _d first order decay constant (h^–1) - k _dw first order decay constant (h^–1) - k _gl rate constant for glucose production (g/l/h) - k _{m, dgr} saturation constant for product degradation (g/l) - k _st rate constant for starch hydrolysis (g/l/h) - k _t1 proportionality constant for amylase production (g mRNA/g substrate) - k _t2 proportionality constant for amylase production (g mRNA *h/g substrate) - k _w protease excretion rate constant (h^–1) - k _wt1 proportionality constant for protease production (g mRNA/g substrate) - k _wt2 proportionality constant for protease production (g mRNA *h/g substrate) - k _wI translation constant (g protease/g mRNA/h) - m maintenance coefficient (g substrate/g cell mass/h) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function, K₁/K₂ less than or equal to 1.0 - Q _w repression function, K₁/K₂ less than or equal to 1.0 - r intrinsic amylase mRNA concentration (g mRNA/g cell mass) - r _m intrinsic protease mRNA concentration (g mRNA/g cell mass) - R _ex retention by the filter of the compounds x=: C starch, E amylase, or S glucose - R _t amylase transport rate (g product/g cell mass/h) - R _wt protease transport rate (g protease/g cell mass/h) - R _s rate of glucose production (g/l/h) - R _c rate of starch hydrolysis (g/l/h) - S ₀ feed concentration of free reducing sugar (g/l) - s extracellular concentration of reducing sugar (g/l) - t time (h) - V volume (1) - w intracellular protease concentration (g/l) - W extracellular protease concentration (g/l) - X cell mass concentration (dry weight) (g/l) - Y yield coefficient (g cell mass/g substrate) - substrate uptake (g substrate/g cell mass/h) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) - _m,dgr maximum specific rate of amylase degradation (h^–1) This study was supported by the Nordic Industrial Foundation Bioprocess Engineering Programme and the Center for Process Biotechnology, The Technical University of Denmark.     

Determination of sulfite radical (SO 3 ⋅ −) reaction rate constants by means of competition kinetics

Summary The sulfite radical anion (SO ₃ ^–) was found to react rapidly with the flavonoid quercetin (k = 2.5 × 10⁸ dm³mol^–1 s^–1) and the carotenoids crocin (k = 1.0 × 10⁹ dm³mol^–1 s^1–) and crocetin (k = 1.5 × 10⁹ dm³mol^1– s^1–). The reactions can easily be monitored due to the strong absorptions of the substrates and, in the case of quercetin, the formation of a strongly absorbing transient species. Using these substances, we determined by means of competition kinetics rate constants of SO ₃ ^– reactions with nucleic acid components, polyunsaturated fatty acids, and glutathione.Abbreviations ABTS 2,2-azinobis(3-ethyl-6-benzothiazolinesulfonate) - cmc critical micellization concentration - GSH Glutathione - PUFA polyunsaturated fatty acids Preliminary results were presented at the Third Biennial Meeting of the Society for Free Radical Research in Düsseldorf in July, 1986     

T-cell receptor-specific monoclonal antibodies against a V β 11-positive mouse T-cell clone

Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a V 11⁺ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the V chain of C6, V 11. This was demonstrated by the fact that the strain distribution pattern of KT11⁺ cells was similar to that of V 5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to V 8 in (B10 × SJL)F₁ × SJL backcross mice. Furthermore, V of C6 has been cloned from a gt10 cDNA library and was demonstrated to be identical to the V 11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11^– cells than E⁺ strains. The KT11⁺ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3-specific antibody 145-2C11.     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Localization of CTβ and C K on mouse chromosome 6

In the mouse three lymphocyte gene families have been positioned on the proximal region of chromosome 6. Originally the immunoglobulin kappa light chain (Igk) and the thymocyte surface antigens Lyt-2 and Lyt-3 were assigned to chromosome 6, and recently the beta chain of the T-cell receptor for antigen was positioned proximal to Igk. Molecular clones which recognize the constant (C region of the beta chain of the T-cell receptor for antigen (C_T ) and the constant region of the immunoglobulin kappa (C _k ) chain were used to determine recombination frequencies with respect to the morphological marker hypodactyly (Hd). SJL/JLw mice were mated with C.B6.C3-Hd/+ mice, and the progeny expressing the Hd phenotype were mated with SJL/JLw mice. Backcross progeny which expressed the Hd phenotype were nephrectomized, and kidney DNA was examined by Southern hybridization for the polymorphic restriction endonuclease fragment (REF) patterns of the parental mice. Of the 88 progeny tested in this three-point cross, 3 C_T and 4 C _K homozygote REF patterns were detected. These homozygotes were mutually exclusive. This implies the following gene order: centromere-C _T -Hd-Igk and C _T1 would be 7.95±2.88 centimorgans from C _K .     

Molecular basis for erythrocyte Le(a+b+) and salivary ABH partial-secretor phenotypes: expression of a FUT2 secretor allele with an A→T mutation at nucleotide 385 correlates with reducedα(1,2) fucosyltransferase activity

TheSe ^wA385T mutation of the FUT2 gene was found to correlate with both the erthrocyte Le(a+b+) and/or salivary ABH partial-secretor phenotypes of Polynesians. Constructs with FUT1 and FUT2 wild type genes, and the FUT2Se ^wA385T,se ^G428A andse ^C571T mutated alleles, were cloned into pcDNAI, and expressed in COS-7 cells. COS-7 cells transfected with theSe ^wA385T allele had weak, but detectable, (1,2)fucosyltransferase activity, with an acceptor substrate pattern similar to the wild type FUT2 gene. Comparative kinetic studies from cell extracts with mutatedSe ^wA385T and wild type FUT2 alleles gave similarK _m values, but less enzyme activity was present in cells transfected withSe ^wA385T (V _max 230 pmol h^–1 mg^–1), as compared to those transfected with FUT2 (V _max 1030 pmol h^–1 mg^–1), suggesting that the mutated enzyme is more unstable. These results confirm that the molecular basis for the erythrocyte Le(a+b+) and the associated ABH salivary partial-secretor phenotype, is an amino acid change of Ile 129Phe in the secretor (1,2)fucosyltransferase.Abbreviations (1,3/1,4)fucosyltransferase GDP-L-fucose:-D-N-acetylglucosaminide 3/4--L-fucosyltransferase - (1,2)fucosyltransferase GDP-L-fucose: -D-galactoside-2--L-fucosyltransferase - bp base pairs - FUT1 H gene; FUT2,Se gene - FUT3 Lewis gene or Fuc-TIll gene - FUT4 Fuc-TIV gene - FUT5 Fuc-TV gene - FUT6 Fuc-TVI gene - MAb monoclonal antibody - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - se ^G428A FUT2 nonsecretor GA mutation at nucleotide 428 - se ^C571T FUT2 nonsecretor CT mutation at nucleotide 571 - Se ^wA385T FUT2 secretor weak AT mutation at nucleotide 385 - SSP sequence specific primer     

Development of reactor model for glucose isomerization catalyzed by whole-cell immobilized glucose isomerase

Based on the kinetic constants determined and the mathematical model of the reactor system developed, the performance of axial flow packed bed continuous enzyme reactor system was studied experimentally and also simulated with the aid of a computer for ultimate objective of optimization of the glucose isomerase reactor system.A reactor model was established analogous to heterogeneous catalytic reactor model taking into account the effect of fluid mass transfer and reversible kinetics. The investigated catalyst system consists of immobilized Streptomyces bambergiensis cells containing the enzyme glucose isomerase, which catalyzes the isomerization of glucose to fructose.List of Symbols A ₀, A ₁, A ₂ parameters in axial dispersion reactor model - c _go, c_g, c_gemol m^–3 glucose concentration at time t=0, at any time and at equilibrium conditions - c _gsmol m^–3 glucose concentration at particle surface - C dimensionless glucose concentration - d _pm particle diameter - d _rm diameter of reactor tube - Da Damkohler number - D _eff m² s^–1 effective glucose diffusion coefficient in Ca-alginate gel beads - k _fm s^–1 film transfer coefficient - K _e equilibrium constant - K _mg, K_mfmol m^–3 Michaelis-Menten constant for glucose and fructose, respectively - K _mmol m^–3 modified Michaelis-Menten constant - K dimensionless parameter - K ^* dimensionless parameter - L m length of reactor tube - Pe Peclet number - Pe _p particle Peclet number - Q m³ s^–1 volumetric flow rate - (-r _g) mol m^–3 s^–1 reaction rate - Re _p Reynolds particle number - Sc Schmidt number - Sh Sherwood number - t s time - v ₀ m s^–1 linear superficial fluid velocity - V _mg, V_mfmol g^–1 s^–1 maximal reaction rate for glucose and fructose, respectively - V _mmol m^–3 s^–1 modified maximal reaction rate for glucose - V _mg ^x mol m^–2 s^–1 maximal reaction rate for glucose - X _g, X_ge glucose conversion and glucose conversion at equilibrium conditions - X normalized conversion - Y dimensionless glucose concentration - void fraction of fixed bed - effectiveness factor of biocatalyst - Pa s kinematic viscosity of substrate - ₁ s first absolute weighted moment - ₂ s² second central weighted moment - _gkg m^–3 substrate density - _pkg m^–3 particle density - ² dimensionless variance of RTD curve - s residence time     

Genetic studies of self-fertility in rye (Secale cereale L.). 2. The search for isozyme marker genes linked to self-incompatibility loci

The segregation of several isozyme marker genes has been studied in F₂ inbred families from hybrids between self-sterile and five self-fertile inbred lines (nos. 2, 3, 4, 5, and 8) as well as from interline hybrids. Self-pollination of F₁ hybrids between self-sterile forms and lines 5 and 8 gave an F₂ segregation ratio of 1 heterozygote:1 homozygote for the gene Prx7 (chromosome 1R) against the allele from the line. This is interpreted as a result of tight linkage of the Prx7 gene with the S1 gene in chromosome 1R (recombination at a level of 0–1%). The self-pollination of such hybrids with lines 2,3 and 4 gave normal segregation for the Prx7 gene (1:2:1). This means that these lines carry a self-fertility allele which is not on chromosome 1R. Interline hybrids 5×2, 5×3 and 5×4 had self-fertility alleles for the two S genes and in inbred F₂ progenies gave the expected deviating segregation for the Prx7 gene in a ratio of 2:3:1. The segregation of interline hybrid 5×8 was normal, 1:2:1, as expected. Highly-deviating segregation in an inbred F₂ family of a hybrid with line 5 has also been obtained for another gene from chromosome 1R — Pgi2 (recombination with the S1 locus of 16.7%). By using the same method it has been estimated that line 4 has a self-fertility allele of the S2 locus from chromosome 2R and that the genes -Glu and Est4/11 are linked with it (recombination 16.7% and 17.5–20% respectively). Lines 2 and 3 have a self-fertility allele of the S5 locus from chromosome 5R which is linked with the Est5-7 gene complex (recombination at a level of 28.8–36.0%).