Adenosine deaminase (ADA) deficiency due to deletion of the ADA gene promoter and first exon by homologous recombination between two Alu elements.

In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Structure and function of the CD7 molecule.

CD7 is a single-domain Ig superfamily molecule expressed on human T and NK cells, as well as on cells in the early stages of T, B, and myeloid cell differentiation. CD7 is highly expressed on malignant immature T cells and is generally absent on malignant mature T cells, such as CD4+ Sezary leukemia and HTLV-1+ adult T-cell leukemia cells. Because of lack of identification of a natural ligand and lack of a monoclonal antibody against murine CD7, the in vivo functions of CD7 have until recently remained obscure. Recent studies in CD7-deficient mice have provided new insights into CD7 function, and demonstrated key roles for CD7 in regulating peripheral T and NK cell cytokine production and sensitivity to LPS-induced shock syndromes. This article reviews recent work on the expression, structure, and function of CD7, and discusses roles the CD7 molecule might play in T and NK cell development and function.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

The c-kit proto-oncogene receptor is expressed on a subset of human CD3-CD4-CD8- (triple-negative) thymocytes.

The c-kit receptor is a tyrosine-kinase transmembrane receptor first identified as an oncogene in the HZ4-feline leukemia virus and later found to be important in hematopoiesis in mice. The ligand for this receptor (Steel factor) can stimulate hematopoiesis both in vitro and in vivo. To study the pattern of c-kit receptor expression in normal human hematopoietic progenitor cells, we prepared a monoclonal antibody (9B9) against human c-kit receptor by using a synthetic peptide (amino acids 476-501) from the extracellular domain of c-kit receptor to immunize Balb/c mice. Monoclonal antibody 9B9 bound to recombinant c-kit protein, the erythroleukemic line HEL, the megakaryocytic line MEG-01, and the murine mast cell line P815. Monoclonal antibody 9B9 also bound to the surface of the CD7+CD3-CD4-CD8- T cell lymphoid cell lines DU.528 and HSB2T, and also to 1 to 4% of normal bone-marrow cells. The majority (67 +/- 6%) of CD34+ bone-marrow progenitor cells coexpressed c-kit receptor. Flow-cytometry analysis of immature CD3-CD4-CD8- (triple-negative) thymocytes indicated 30 +/- 9.5% expressed the c-kit receptor, and thymidine incorporation assay revealed that the receptor is functional. Indirect fluorescent microscopy of human thymic tissue, using a monoclonal antibody against Steel factor, revealed its presence on scattered mononuclear cells within the intralobular septae and the subcapsular cortex, which are regions where the triple-negative thymocytes are also localized. These data provide evidence that the c-kit receptor is present on human hematopoietic bone marrow and intrathymic T cell progenitor cells, and that it likely plays a role in early T cell lymphopoiesis.

Isolation and characterization of a human pro alpha 2(I) collagen gene segment.

Over 20 kilobase pairs of the human pro alpha 2(I) collagen gene have been isolated and characterized by restriction endonuclease mapping, cell-free translation of hybrid-selected RNA, and DNA sequence analysis. We have sequenced an exon and determined its length to be 108 base pairs (bp). This is consistent with the organization of chick and sheep collagen genes in that exons are multiples of 9 bp in length, frequently being 54 and 108 bp. The sequenced exon was bordered by a GT (guanine-thymine) at its 3' end and an AT (adenine-thymine) at its 5' end. This pattern has been found at all normal intron-exon junctions in eukaryotic cells. The amino acid sequence derived from DNA sequencing of this 108 bp exon revealed 88% homology compared to the amino acid sequence of bovine pro alpha 2(I). The bases encoded 12 Gly-X-Y triplets characteristic of the helical portion of collagen. A unique sequence Gly-Gly-Lys-Gly-Glu-Lys identified this fragment as alpha 2(I) collagen.

Comparison of human and Xenopus GATA-2 promoters.

GATA proteins comprise a family of transcription factors that are required for appropriate development of hematopoietic lineages. In order to understand the transcriptional regulation of GATA genes, we have cloned the human GATA-2 gene and identified and characterized its promoter. Comparison with the Xenopus GATA-2 promoter demonstrates highly conserved CCAAT box elements, which are essential for appropriate Xenopus expression. Unlike the Xenopus gene, the human GATA-2 gene lacks GATA binding motifs within the first 800 bp of 5' flanking sequence. In addition, the human GATA-2 promoter has two highly conserved ets sites that resemble the binding site for a recently described ets repressor factor, ERF. These conserved DNA sequence motifs provide strong candidate regions for the regulation of GATA-2.

Genotypic divergence precedes clinical dissemination in a case of synchronous bilateral B-cell malignant lymphoma of the testes.

Malignant lymphoma of the testis occurs bilaterally more often than any other tumor type. We report the case of a 62-year-old man who presented with synchronous, bilateral, testicular malignant lymphomas without clinical or radiologic evidence of extratesticular disease. The patient received no therapy other than bilateral orchiectomy and subsequently developed widespread disease 6 months later. Southern blot DNA analysis was performed on the initial orchiectomy samples for immunoglobulin (Ig) gene rearrangements. These genotypic analyses showed different clonal rearrangements in the Ig heavy chain JH region but identical clonal rearrangements in the Ig light chain C Kappa region. To our knowledge this is the first genotypic demonstration of a common clonal origin in synchronous, bilateral, testicular malignant lymphomas. We interpret these findings as molecular evidence that the patient's malignant lymphoma was already disseminated at initial presentation, although it was clinically undetectable at that time.

Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain.

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

Gamma-globin gene promoter elements required for interaction with globin enhancers.

Normal expression of the human beta-globin domain genes is dependent on at least three types of regulatory elements located within the beta-globin domain: the locus control region (LCR), globin enhancer elements (3'beta and 3'Agamma), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated gamma-globin promoters linked to the 5'HS2 enhancer. We show that in K562 cells, 5'HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 --> +25) gamma promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5'HS2. Mutation of the gamma-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5'HS2. To determine if enhanced expression of gamma-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH gamma-globin gene promoters to 5'HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Agamma-globin gene 3' enhancer to a plasmid containing the gamma-globin gene promoter and 5'HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5'HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5', but not 3', to the gamma-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the gamma-globin promoter and the LCR/5'HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

An analysis of alternatively spliced CD45 mRNA transcripts during T cell maturation in humans.

CD45 is a transmembrane protein tyrosine phosphatase found on nucleated hematopoietic cells. In humans, multiple protein isoforms of CD45 are produced by alternative mRNA splicing of exons 4, 5, and 6 coding for the extracellular portion. We measured all eight possible CD45 mRNA transcripts using RT-PCR in human thymocytes and T cell lines. We report that only six mRNA transcripts are present in T cells. The high mw CD45 mRNA transcripts containing exon 4 correlated with the stage of T cell maturation: abundant high mw transcripts (30.7% of all CD45 mRNA transcripts) were present in immature, CD3-4-8 triple-negative thymocytes which decreased (7.7%) in intermediate, CD4+8+ double-positive (DP) thymocytes and then increased (13.8% or 16.8%) in mature, CD4+8- or CD4-8+ single-positive thymocytes. In addition, there was a complex variation in the spliced mRNA transcripts coding for CD45R(O), CD45R(B), CD45R(BC), CD45R(AB), and CD45R(ABC) protein isoforms. High mw CD45 mRNA transcripts accumulated immediately prior to an important physiologic event such as thymocyte expansion. In addition, we identified linkage between RNA splicing of exons 5 and 6, and splicing of exon 5 only and exons 4, 5, and 6 in FACS-purified CD4+ and CD8+ thymocytes. These data support the developmental regulation of alternatively spliced CD45 mRNA transcripts and suggest that specific CD45 isoforms may play an important role at critical stages of T cell development.

Analysis of globin gene structure in patients with beta thalassemia by restriction endonuclease mapping.

Twenty-six DNA samples from individuals either heterozygous or homozygous for beta thalassemia were analyzed by restriction endonuclease digestion, agarose gel electrophoresis, and Southern blot analysis to define DNA fragments containing portions or all of the beta globin gene. A total of twenty-seven genes affected by a beta thalassemia mutation and twenty-seven genes affected by a beta thalassemia mutation and twenty-two normal beta globin genes were examined in Italian, Greek, or Asian individuals. With all four restriction endonucleases used, the fragments generated from DNA of thalassemic individuals were identical to those found in DNA from normal. Thus, gross rearrangement or deletion within the genomic region containing the beta globin gene is not characteristic of mutations which cause a thalassemia. A third patient homozygous for pancellular hereditary persistence of fetal hemoglobin was shown to have complete deletion of the delta and beta globin genes.

Dissociation of thymidine incorporation and transferrin receptor expression from cell growth and c-myc accumulation in alpha-interferon-treated cells.

Alpha-interferon is capable of altering the pattern of growth of both normal and neoplastic cells, but the pathways essential to sensitivity and resistance to alpha-interferon are unknown. To explore the growth inhibition induced by alpha-interferon, we examined the interferon-sensitive cell line Daudi and the resistant cell line HL-60. In Daudi, alpha-interferon induced a fall in c-myc mRNA accumulation at 24 h, inhibited tritiated thymidine ([3H]Thd) uptake at 48-72 h, and inhibited proliferation at 72-96 h. The half-life of c-myc mRNA was shortened from 31 to 13 min by alpha-interferon treatment. In HL-60, no alteration in c-myc accumulation or cell growth was observed, but [3H]Thd uptake was inhibited by 49%. Exogenous thymidine partially reversed the effects of alpha-interferon on [3H]Thd incorporation. The number of transferrin receptors, as measured by immunofluorescence, was unaffected by alpha-interferon in both cell lines. We conclude that the growth inhibitory effects of alpha-interferon are neither dependent upon inhibition of thymidine metabolism nor on expression of the transferrin receptor, but may be linked to control of c-myc.

Multiple red cell ferritin mRNAs, which code for an abundant protein in the embryonic cell type, analyzed by cDNA sequence and by primer extension of the 5'-untranslated regions.

Ferritin maintains iron in a bioavailable, nontoxic form for vertebrates and invertebrates, higher plants, fungi, and bacteria; the protein is formed from two classes of subunits (H and L) in ratios which vary in different cell types. Ferritin may be an abundant, differentiation-specific protein or a "housekeeping" protein. The red cells of embryos are specialized for iron storage and have abundant ferritin; iron regulates the synthesis of ferritin in such cells translationally by recruitment of stored, ferritin mRNA and by translational competition. To characterize mRNA regulated in such a manner, we prepared cDNA from reticulocytes of bullfrog tadpoles, a readily available source of embryonic red cells; moreover, no protein sequence information was available for nonmammalian ferritin. An almost full-length (817 base pairs) cDNA (pJD5F12) was isolated and sequenced, the 5' end was analyzed by primer extension, and the cloned DNA was used as a hybridization probe. We have shown that ferritin mRNA is stored in the cytoplasm and that the 5' end of the mRNA is heterogeneous. The 5'-untranslated region of ferritin mRNA consisted of 143 nucleotides in the major (65%) species and 146 or 152 in the minor species (approximately 17% each). (Heterogeneity is characteristic of some other abundant mRNAs, e.g. globin, which is also translationally regulated.) Since excess iron had no detectable effect on the heterogeneity of the 5' end of ferritin mRNA, the feature is more likely associated with mRNA abundance and/or cell specialization than translational control. In the bullfrog, as in humans and rats, ferritin is encoded by multiple genomic sequences (four to eight) which specify proteins of considerable homology. For example, 75 of the 81 amino acids present in all mammalian ferritins sequenced are also present in the frog; the overall homology between frogs and humans or rats is 59-66%. Ferritin H and L subunits in humans are distinct (overall homology 56%) and appear to have diverged from a common precursor relatively recently. In contrast, ferritin H and L subunits have high homology in tadpole red cells, determined by hybrid select translation, which suggests that bullfrog red cell ferritin may be close to the primordial sequence.

Dibutyryl cyclic adenosine monophosphate reduces expression of c-myc during HL-60 differentiation.

The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo-cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP-dependent protein kinase is reviewed.

Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle.

Increased contractile activity of skeletal muscle augments the volume fraction and enzymatic capacity of mitochondria and suppresses the enzymatic capacity of several cytoplasmic enzymes of glycolysis. To examine the biochemical mechanisms underlying these effects, we measured the concentrations of cytochrome b mRNA and aldolase A mRNA in tibialis anterior muscles of adult rabbits that had been stimulated via the motor nerve to contract continuously at 10 Hz for 5 or 21 days; these were compared with the corresponding levels in the unstimulated limbs of the same animals. After 21 days of stimulation aldolase mRNA had fallen to one-fourth of control levels, while cytochrome b mRNA had increased by 5-fold. A reduction in aldolase mRNA was already evident after only 5 days of stimulation, whereas the level of cytochrome b mRNA was not elevated at this stage. Mitochondrial DNA was unchanged after 5 days but had increased by 4-fold after 21 days. We conclude that contractile activity in skeletal muscle produces reciprocal changes in the expression of these two genes at a transcriptional level but via different regulatory mechanisms. Enhancement of the expression of the mitochondrial cytochrome b gene appears to be proportional to the increase in its copy number and may not, therefore, depend upon changes in transcriptional or translational efficiency. The reduction in aldolase A mRNA occurs at an earlier stage in the response to contractile activity and is probably mediated by a reduced transcriptional efficiency.