Cloning and expression of the rat class I MHC gene RT1.A l

Correspondence to: T. J. Gill, III.     

Cytotoxic T lymphocytes of the rat are predominantly restricted by RTL1.A and Not RT1.C-determined major histocompatibility class I antigens

Two types of biochemically defined class I major histocompatibility complex (MHC) antigens are found in the rat, RTLA antigens that are ubiquitously expressed and RTLC antigens which so far are detectable only on certain cell types, notably B and T lymphocytes. It is shown that the cytotoxic T lymphocyte response to minor H antigens of the LEW strain, including the H-Y antigen, and to TNP-modified syngeneic lymphoid cells is restricted by RTLA but not RTLC gene products. This conclusion is based on bulk culture assays including cold target inhibition tests and limiting dilution experiments using recombinants between the RT1 ^a and RT1 ^u haplotypes. The possibility that class I MHC antigens exist which have no major restriction function is discussed.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.     

Water-soluble form of RT1.A class I MHC molecules in the kidney and liver of the rat

The RT1.A (H-2K,D type) class I major histocompatibility complex (MHC) antigens of the rat are well recognized as membrane-bound glycoproteins. In this report, we demonstrate that liver and kidney in the DA rat strain contain large amounts of a water-soluble RTl. A class I molecule with a discrete heavy chain approximately 5 kd smaller than the membrane-bound form. An identical molecule could be identified in DA rat serum. This small class I molecule carries all of the polymorphic antigenic determinants of the RT1.A^av1 class I molecule. The water-soluble molecule is readily denatured in its pure form when frozen and thawed, but this does not occur when it is mixed with serum, presumably because of a stabilizing interaction with one or more carrier proteins. The half-life of the class I molecule in serum was measured to be approximately 1.5 h. The LEW rat strain produced detectable but substantially smaller amounts of water-soluble RT1.A molecules. Our studies indicate that RT1.A^av1 class I MHC antigens are synthesized and presumably secreted in a smaller water-soluble form by liver, kidney, and possibly other tissues under physiological conditions, a point of con-siderable interest in view of the immunoregulatory functions of the membrane-bound forms of these molecules.     

Sequence analysis of the promoter regions of the classical class I gene RT1.A l and two other class I genes of the rat MHC

The nucleotide sequence data reported in this Papershave been submitted to the GenBank, EMBL, and DDBJ nucleotide sequence databases and have been assigned the accession numbers X79719 (RT1.A ¹), X79720 (RT1.C ¹), and X79721 (RT12.5)     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

A simplified procedure for the purification of rat liver tyrosine aminotransferase.

Rat liver tyrosine aminotransferase was purified by chromatography on CM-Sephadex C-50 and DEAE-cellulose, (NH4)2SO4 fractionation and gel filtration on Sephadex G-200. Livers from 400 rats can be easily worked up by this procedure. Furthermore, this purification method has the advantage that hepatic tryptophan 2,3-dioxygenase, which, like tyrosine aminotransferase, is induced by glucocorticosteroids, can be purified from the same homogenate. Tyrosine aminotransferase purified by this method was shown to be specific for 2-oxoglutarate. Its subunits have a molecular weight of 45 000. The following "apparent" Michaelis constants were determined: L-tyrosine, 1.7 X 10(-3) M; 2-oxoglutarate, 5.9 X 10(-4) M; and pyridoxal 5'-phosphate, 2.1 X 10(-6) M. Tyrosine aminotransferase, depleted of its cofactors, binds 4 molecules of pyridoxal 5'-phosphate per 90 000 daltons with a KA of 2.2 X 10(5) M-1.     

An affinity-column procedure for the purification of veratrate O-demethylase from fungi.

An affinity column procedure is reported for purifying veratrate O-demethylase from higher fungi. The procedure is based on the affinity of the fungal demethylases for veratrate, which was coupled to AH-Sepharose 4B. An over 300-fold purification of the enzyme from an Ascomycete (Chaetomium piluliferum), and a lower degree of purification (20-fold) from a Basidiomycete (Xerocomus badius), were obtained. The O-demethylases from higher fungi require NADH and oxygen. The enzyme activity is sensitive to exposure to oxygen. The pH optima are 5 for enzyme from Chaetomium, and 7 for demethylase from Xerocomus, respectively. The enzymes are not specific for veratrate. They also demethylate p- and m-anisate and 3,4-dimethoxycinnamate, but to a lower degree.