Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by α1 and α2 but not α3 external domains

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2 ^dmice use H-2L^d but not H-2D^d or H-2K^d antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2L^d protein recognized by CTL, we constructed a recombinant H-2L ^d/D ^dgene encoding a hybrid antigen with 1 and 2 external domains of H-2L^d and 3, transmembrane and cytoplasmic domains of H-2D^d. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2L^d/D^d molecules were recognized by LCMV- and VSV-specific H-2L^d-restricted CTL in a manner similar to that of wild-type H-2L^d antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the 3 domain of H-2L^d fused to 1 and 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2L^d-restricted CTL directed against LCMV and VSV recognize determinants controlled by the 1 and/or 2 domains of the H-2L^d molecule.Abbreviations used in this paper CTL cytotoxic T lymphocytes - VSV vesicular stomatitis virus - LCMV lymphocytic choriomeningitis virus - tk thymidine kinase - HAT hypoxanthine, aminopterine, thymidine - HSV herpes simplex virus - FCS fetal calf serum - SAC Staphylococcus aureus Cowan I strain - TM transmembrane - CYT cytoplasmic     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Cell-surface expression of H-2Db requires N-linked glycans

The question of whether beta-2 microglobulin (B2m)-independent expression of the mouse major histocompatibility complex (MHC) antigen H-2D^b results from the atypical glycosylation pattern associated with this MHC antigen (i. e., three glycans instead of two) has been addressed. Cell-surface expression of transfected H-2D^b in the B2m deficient cell line RIE was completely abolished by the drug tunicamycin (Tm). Introduction of a functional B2m gene by transfection did not re-establish cell-surface expression of D^b in the presence of Tm. Tm had no effect, however, on the expression of a truncated D^b molecule lacking the 1 and 2 domains which is glycosylated at amino acid position 256, suggesting that the D^b molecule, unlike other class I antigens, possesses an unstable conformation in the 1 and/or 2 domains which requires the attachment of glycans before it is transported to the cell surface. Once attached, however, glycans may confer a stable 1/2 conformation apparently peculiar to D^b which allows cell-surface expression in the absence of B2m.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Purification, cloning, and properties of α-galactosidase from Saccharopolyspora erythraea and its use as a reporter system

An -galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K_m for p-nitrophenyl--d-glucopyranoside (pNPG), K_m for melibiose and the V_max are similar to those of other studied -galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related -galactosidase enzymes. This fragment was used as a probe to clone the -galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique -galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The -galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl--d-galactosidase (X--gal) on agar media and pNPG in liquid media.     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

A new α2HS-glycoprotein typing by isoelectric focusing

Summary Isoelectric focusing (pH 4.0–5.0) of serum ₂HS-glycoprotein on polyacrylamide gels has been found to be a useful tool in population genetics and forensic science. Using this method, we isolated three common types, ₂HS 1-1, ₂HS 2-1 and ₂HS 2-2, and showed that ₂HS types are determined by two autosomal codominant alleles, ₂ HS ¹ and ₂ HS ². The method is simple, fast and easy to perform. Results of typing for the two alleles, ₂ HS ¹ and ₂ HS ², are described for a Japanese population sample (n=1003).     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Mikrochemische Untersuchung der α-d-Glucosidasen mit 4-Methylumbelliferyl-und 2-Naphthyl-α-d-glucosid

Zusammenfassung In Rohhomogenaten aus gefriergetrockneten Kryostat-schnitten von verschiedenen Rattenorganen werden die K _m und V _max der neutralen und sauren -d-Glucosidase bestimmt und der Einfluß von pH, Substrat- und Enzymkonzentration und Inkubationszeit auf die Aktivität fluorometrisch mit 4-Methylumbelliferyl-und 2-Naphthyl--d-glucosid als Substraten ermittelt.Mit den biochemischen Daten werden 2 mikrochemische Ansätze zur fluorometrischen Messung dieser Glykosidasen entwickelt und die saure und neutrale -Glucosidase in Gruppen von Epithelzellen nach Isolierung aus gefriergetrockneten Kryostatschnitten von Nebenhoden, Jejunum, Ilium, Niere und Leber untersucht. Im Vergleich zum 2-Naphthylderivat sind beide -Glucosidasen mit 4-Methylumbelliferyl--d-glucosid weniger aktiv. Allerdings fluoresziert 4-Methylumbelliferon etwa 100mal intensiver als 2-Naphthol, so daß das Methylumbelliferonderivat zur Messung der -Glucosidasen speziell in schwach aktiven Zellen der 2-Naphthylverbindung vorzuziehen ist.

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Microchemical investigation of -d-glucosidases using 4-methylumbelliferyl-and 2-naphthyl--d-glucoside

Summary In crude homogenates prepared from freeze-dried cryostate sections of various rat organs the K _m and V _max of acid and neutral -glucosidase as well as the effect of the pH, substrate and enzyme concentration and the incubation time on the activity were determined fluorometrically with 4-methylumbelliferyl-and 2-naphthyl -d-glucoside as substrates.On the basis of the biochemical data 2 assays were developed for the microchemical measurement of both -glucosidases in groups of epithelial cells isolated from freeze-dried cryostate sections of the epididymis, jejunum, ilium, liver and kidney of suckling and adult rats. The rate of hydrolysis of 2-naphthyl and 4-methylumbelliferyl -d-glucoside differs moderately. However, due to the higher sensitivity of 4-methylumbelliferone the methylumbelliferyl derivative is preferable especially for the evaluation of -d-glucosidases in cells with low enzyme activity.

     

Biochemical documentation of allelic variation of the I-E antigens of the d,k, p,r, and u haplotypes

The extent of allelic variation of the E and E polypeptide chains of the I-E antigens from the H-2> ^d ,H-2 ^k , H-2 ^p , H-2 ^r , and H-2 ^u haplotypes is described. E and E chains were individually labeled with arginine or lysine and compared by tryptic peptide analysis. The results indicate minimum variability among the E polypeptides encoded by the d, k, p, and r haplotypes. However, the E ^u chain differed significantly from the other allelic E gene products. On the other hand, the E alleles demonstrated substantial variability with the E ^d being notably less similar to the other alleles than they are to each other. These findings are consistent with a number of observations regarding the serology and functions of the I-E antigens.Abbreviations MHC major histocompatibility complex - NMS normal mouse serum - NP-40 Nonidet P-40 - NTS 0.25% NP-40, 10 mM Tris-Cl, 0.15 M NaCl (pH 7.4) - SDS sodium dodecylsulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS     

Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

H-2U: A new region at the D end of the murine MHC

A new genetic region, mapping within the H-2 complex, has been serologically defined with several alloantisera raised in mice which differ at the D region. When these antisera were absorbed to remove H-2D antibodies, residual antibody activity remained that reacted in a strain-specific manner, and the antigens involved mapped to a new genetic region between the S and D regions. Two allelic variants relating to the d and k haplotypes have been defined by genetic mapping studies. This new region has been designated H-2U and the antigens it controls appear to resemble Ia antigens in their cellular distribution and molecular weight. The new antigen is primarily expressed on B cells, and is carried on protein molecules having approximate molecular weights of 36 000 and 60 000 daltons and resembling the and - chain dimer characteristic of Ia antigens.     

Evolution of the single copyαA-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3′ non-coding regions

A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat A₂-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3-untranslated regions of A₂-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The A₂-mRNA 3-non-coding regions of reptiles and birds are 300–550 bases longer than those of mammals. Some rodents produce next to the A₂-mRNA another messenger that encodes the A^lns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the A₂-polypeptide chain. A₂ and A^lns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides foundin vivo andin vitro. The size heterogeneity of the A₂-mRNA from most mammals examined is due to the variable size of the poly(A) tail.     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.