Cultured cells depend on cytokine mediators for sustained growth and maintenance and are routinely employed in bioassays to detect and measure minute changes in biological mediators, e.g. the interferons and interleukins. We evaluated the effects of mycoplasma infection on the steady-state mRNA levels of two cytokines IL-1 alpha and beta. Noninfected human squamous carcinoma cell line A431 expressed constitutively IL-1 alpha and beta mRNA. In contrast freshly isolated peripheral blood mononuclear cells and the monocytic cell line U937 expressed abundant IL-1 mRNA only after the appropriate stimulation. Peripheral blood mononuclear cells and U937 steady-state IL-1 beta mRNA levels were considerably greater than IL-1 alpha mRNA levels, whereas nearly equivalent high levels of IL-1 alpha and beta mRNA were detected in A431 cells. Mycoplasma infection of cultured A431 cells reduced the steady-state levels of IL-1 alpha and beta mRNA. This effect was nonspecific for A431 cells as actin mRNA steady-state levels showed similar decreases to mycoplasma contamination. However, this response was cell specific since mycoplasma-free and contaminated U937 cells differed little in IL-1 mRNA expression. These results show that the response to mycoplasma infection is at least partly cell-type dependent.
Mouse myeloma ribosomes were prepared without exposure to detergent and their behavior with either decreasing concentrations of Mg 2+ or increasing concentrations of KCl examined. The minimal conditions for dissociation into subunits were determined and the properties of the subunits obtained under different conditions compared. Ribosomes which are first freed of nascent chains dissociate into 40 S and 60 S subunits at physiological Mg 2+ concentrations (5 mM) and at only 0.3 M KCl (K3 subunits). Although they lack one distinct protein present in low Mg 2+ subunits, these K3 subunits reassociate spontaneously to form 80 S particles at lower ionic strength (0.05 M KCl) and are highly active in poly(U)‐directed polyphenylalanine synthesis. This activity is maintained after storage at—70 °C for several months. Subunits obtained at a higher concentration of KCl (0.88 M) are less active and less stable, and they lack at least one protein which is present in K3 subunits. Low Mg 2+ subunits are unable to reassociate spontaneously, are inactive in the poly(U) system, have lower sedimentation coefficients, and have lost the 5 S RNA.
The 14S mRNA from MOPC 173 tumour has been transcribed into cDNA by AMV DNA polymerase and converted into a double stranded form by Escherichia coli DNA polymerase I. After addition of oligo-dG tracts at the 3'-OH ends by calf thymus terminal transferase this DNA was hybridized to an E. coli plasmid (pCR1) to which oligo-dG tracts had been similarly added. Circular molecules resulting from GC base pairing have been used to transform C 600 E. coli cells and to confer kanamycine resistance. Several recombinants have been obtained containing the V+C regions and the C or V region alone. These recombinant molecules are being used to analyse the translocation of V and C genes and to purify V and C genes from DNA of germ line cells and of differentiated tumour.
Recent progress in the molecular genetics of HLA class II antigens has revealed the existence of multiple loci and of a large degree of polymorphism, with more individual alleles than was expected. An accurate detection and analysis of this extensive polymorphism is essential for optimal HLA typing for transplantation and for a reevaluation of HLA-disease association. Because of the limitations of the current typing methods, including restriction fragment length polymorphisms, we have proposed a DNA typing procedure based on hybridization with loci- and allele-specific oligonucleotides. Here we present a much simpler way of analyzing class II micropolymorphism down to the level of single nucleotide differences. RNA oligonucleotide typing (ROT) relies on RNA dot blots and requires 10-20 ml of blood. It is shown that with appropriate oligonucleotide probes, ROT can reliably and unambiguously identify any polymorphism at any of the HLA loci, including new alleles, not identified with previous methods. This illustrates the importance of oligonucleotide typing to optimize HLA matching, in particular for transplantation involving unrelated donors.
Major histocompatibility complex (MHC) class II deficiency, or bare lymphocyte syndrome (BLS), is a disease of gene regulation. Patients with BLS have been classified into at least three complementation groups (A, B, and C) believed to correspond to three distinct MHC class II regulatory genes. The elucidation of the molecular basis for this disease will thus clarify the mechanisms controlling the complex regulation of MHC class II genes. Complementation groups B and C are characterized by a lack of binding of RFX, a nuclear protein that normally binds specifically to the X box cis-acting element present in the promoters of all MHC class II genes. We have now purified RFX to near homogeneity by affinity chromatography. Using an in vitro transcription system based on the HLA-DRA promoter, we show here that extracts from RFX-deficient cells from patients with BLS (BLS cells) in groups B and C, which are transcriptionally inactive in this assay, can be complemented to full transcriptional activity by the purified RFX. As expected, purified RFX also restores a completely normal pattern of X box-binding complexes in these mutant extracts. This provides the first direct functional evidence that RFX is an activator of MHC class II gene transcription and that its absence is indeed responsible for the regulatory defect in MHC class II gene expression in patients with BLS.
In renal transplantation a good HLA-DR match is associated with a higher success rate of graft outcome. However, due to a number of technical problems, reliable serological DR typing cannot always be obtained, and the very large number of HLA-DR alleles now discovered renders such DR matching more difficult. In view of the medical importance of HLA class II polymorphism in transplantation immunology, we have developed a simple HLA-DR oligotyping procedure on PCR-amplified DNA, by hybridization with 14 synthetic oligonucleotide probes able to recognize all major HLA-DR specificities. In particular, the probes used in this study allow the unambiguous discrimination of the DRw11, w12, w13, w14-Dw9 specificities or of rare alleles such as DR-Br or DRw13-DwHAG, which are very often difficult or impossible to identify by serology. In order to explore the potential of this methodology, we have analyzed by oligotyping 110 kidney transplant patients with doubtful or unreliable serological assignment, or with DR blank alleles. Comparison between serology and oligotyping shows that in 66.3% of the patients we observed an excellent correlation. About half these patients are homozygotes, as ultimately verified by RFLP typing. In 26.4% of the patients however, at least one HLA-DR antigen was discrepant, and in 7.3% of the cases oligotyping resolved uninterpretable serology. Almost all of the discrepancies were due to errors in allele assignment within the DRw52 group, mostly in the case of DRw13 alleles. This study confirms the expected qualitative advantage of the oligotyping technique and its simplicity as compared with the RFLP DNA typing procedure. Large scale application of the oligotyping methodology will therefore be beneficial to optimize HLA-DR matching in organ transplantation, particularly in high responders with first kidney graft rejection.
