Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown.

Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis.

The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors. We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3. These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes. Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes. HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other. Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1. We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter. However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences. Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription. Alternatively, TF-X1 may not be a DNA-binding protein at all.

Transcription and replication of human immunodeficiency virus-1 in B lymphocytes in vitro.

We and others have shown that HIV-1 transcription and replication in vitro are increased by the binding of transcriptional enhancer DNA sequences in the HIV-1 long terminal repeat (LTR) to a cellular protein designated Nuclear Factor-kappa B (NF-kappa B), a trans-acting transcription factor which is present in activated but not in resting T cells. Because NF-kappa B is also expressed in resting B cells we suggested that B cells might provide an optimal intracellular environment for HIV-1 transcription. Using a transient transfection assay, we show that the HIV-1 LTR is indeed more efficiently transcribed in the B cell line Raji than in the T cell line Jurkat, and that this effect maps to the transcriptional enhancer (NF-kappa B binding site) of the LTR. Furthermore, we show that Raji cells which have been rendered CD4-positive by gene transfection are susceptible to HIV-1 infection, and that viral gene expression and replication are more efficient in these CD4-positive B cells than in CD4-positive T cells. These findings demonstrate the crucial role of the transcriptional enhancer in regulating HIV-1 expression and, since receptors for HIV-1 are expressed by some Epstein-Barr virus (EBV)-positive B cells, they also suggest that HIV-1 replication could occur in EBV-positive B cells in vivo.

Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product.

Human immunodeficiency virus-1 (HIV-1) gene expression is controlled by cellular transcription factors and by virally encoded trans-activation proteins of the HIV-1 tat and art/trs genes, which are essential for viral replication. Tat trans-activates HIV-1 gene expression by interacting with the trans-acting response element (TAR) located within the HIV-1 long terminal repeat (LTR) (ref. 2). In transient expression assays, tat mediates its effects largely by increasing the steady-state levels of messenger RNA species that contain the TAR sequence at or near their 5' ends, suggesting a function for tat either in transcription or in subsequent RNA processing. The tat gene could also facilitate translation of mRNA containing the TAR sequence. To determine the mechanism of trans-activation by tat, we analysed the structure and rate of synthesis of RNA species directed by the HIV-1 LTR in transient expression assays both in the presence and absence of tat. Although the rate of HIV-1 transcription initiation was not affected by tat, transcriptional elongation beyond position +59 was seen only in the presence of tat. Thus, tat trans-activates HIV-1 transcription by relieving a specific block to transcriptional elongation within the TAR sequence.

Mutant human B cell lines deficient in class II major histocompatibility complex transcription.

By mutagenesis and selection, two independent Ia- mutant cell lines derived from the Ia+ human B cell line Raji were isolated. One mutant failed to express detectable surface HLA-DR and HLA-DQ molecules, while the other expressed very low levels of these molecules. mRNA encoding Ia molecules was correspondingly absent or reduced in these cell lines. In contrast, levels of class I major histocompatibility complex (MHC) mRNA and protein were normal in these cells. The class II MHC genes of the mutant cells were grossly intact, suggesting that the defective expression of these genes was due to an absent or abnormal trans-acting regulatory factor. In vitro nuclear run-on transcription assays and measurement of class II MHC mRNA stability suggested that the specific defect in class II MHC gene expression in both mutant cell lines was at the level of transcription initiation.

A model for the transcriptional regulation of MHC class II genes.

The analysis of la antigens has focused primarily on their structure and function. However, the quantitative differences in immune response gene expression are equally important, since variation of cell surface la influences immunoregulation and the expression of immunologically mediated disease. Here, Kate Sullivan and her colleagues review the current knowledge of and propose a model for the transcriptional regulation of immune response gene expression.

Expression of T cell receptor genes in human B cells.

We analyzed the transcription and rearrangement of the T cell antigen receptor (Ti) genes Ti alpha and Ti beta in human B cell, T cell, and myeloid cell lines, as well as in purified tonsillar B and T cells. All four B cell lines examined, as well as one of two myeloid cell lines, expressed low levels of truncated Ti beta transcripts, as did freshly purified tonsillar B cells. Two of the B cell lines and one of the myeloid lines also expressed truncated Ti alpha transcripts, while tonsillar B cells did not. Sequence analysis of cDNA clones from a B cell line demonstrated that these truncated Ti alpha and Ti beta transcripts were composed of unrearranged J and C gene segments. Comparison of cDNA clones from T and B cells suggests that D alpha genes or N regions contribute to the formation of Ti alpha transcripts in T cells but not in B cells. None of the B cell or myeloid cell lines in this study showed evidence of Ti beta gene rearrangements by Southern blotting. Our data, and other studies of gene rearrangements in human tumors, demonstrate that the level of Ti beta transcriptional activity and the frequency of Ti beta gene rearrangements are correlated in all cell types examined. Thus, our data support the accessibility model of antigen receptor gene rearrangement, whereby the susceptibility of gene segments to recombination enzymes is correlated with their transcriptional activity.

Reconstitution of an active surface T3/T-cell antigen receptor by DNA transfer.

We have introduced a full-length complementary DNA of the T-cell antigen receptor beta-chain into a mutant human T-cell line that lacked a complete beta-chain messenger RNA, had a diminished level of alpha-chain transcript and did not express surface T3- or antigen receptor. Expression of the transfected beta-chain led to a normal level of alpha-chain transcript and a structurally and functionally active T3 T-cell antigen receptor complex on the cell surface.