Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.

Usher syndrome type IIa (OMIM 276901), an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa, maps to the long arm of human chromosome 1q41 between markers AFM268ZD1 and AFM144XF2. Three biologically important mutations in Usher syndrome type IIa patients were identified in a gene (USH2A) isolated from this critical region. The USH2A gene encodes a protein with a predicted size of 171.5 kilodaltons that has laminin epidermal growth factor and fibronectin type III motifs; these motifs are most commonly observed in proteins comprising components of the basal lamina and extracellular matrixes and in cell adhesion molecules.

Genotype-phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations.

BACKGROUND: PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype-phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide. PATIENTS AND METHODS: Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed. RESULTS: The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G-->A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%), 5% in 4 (6%), "reduced" in 2 (3%) (not reported, n = 54). Nonsense mutations were significantly associated with younger age at onset (p<0.001) and absent natural killer activity (p = 0.008). CONCLUSION: PRF1 mutations are spread over the functional domains. Specific mutations are strongly associated with Turkish, African American and Japanese ethnic groups. Later onset and residual cytotoxic function are observed in patients with at least one missense mutation.

Structure and expression of B-myc, a new member of the myc gene family.

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.

6;7 chromosomal translocation in spontaneously arising rat immunocytomas: evidence for c-myc breakpoint clustering and correlation between isotypic expression and the c-myc target.

Our previous studies have shown that spontaneously arising immunocytomas in the LOU/Ws1 strain of rats contain a t(6;7) chromosomal translocation in all seven tumors studied (F. M. Babonits, J. Spira, G. Klein, and H. Bazin, Int. J. Cancer 29:431-437, 1982). We have also shown that the c-myc is located on chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature (London) 306:497-499, 1983) and the immunoglobulin H cluster on chromosome 6 (W.S. Pear, G. Wahlström, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Immunogenetics 23:393-395, 1986). We now report a detailed cytogenetic and molecular analysis of nine additional rat immunocytomas. The t(6;7) chromosomal translocation is found in all tumors. Mapping of the c-myc breakpoints showed that in 10 of 14 tumors, the c-myc breakpoints are clustered in a 1.5-kilobase region upstream of exon 1. In contrast with sporadic Burkitt's lymphoma and mouse plasmacytoma, only 1 of 14 tumors contains the c-myc breakpoints in either exon 1 or intron 1. Analysis of the sequences juxtaposed to the c-myc show that immunoglobulin H switch regions are the targets in at least five tumors and that there is a strong correlation between the secreted immunoglobulin and the c-myc target. Unlike sporadic Burkitt's lymphoma and mouse plasmacytoma, at least two rat immunocytomas show recombination of the c-myc with sequences distinct from immunoglobulin switch regions.

The gene for a novel transmembrane protein containing epidermal growth factor and follistatin domains is frequently hypermethylated in human tumor cells.

A DNA fragment frequently hypermethylated in tumor cells was isolated using a novel screening strategy termed methylation-sensitive arbitrarily primed PCR. The isolated sequence corresponded to a CpG island at the 5' end of a previously unknown gene, TPEF (transmembrane protein containing epidermal growth factor and follistatin domains). Expression of TPEF was observed using Northern master blot analysis of a variety of normal tissues including colon, bladder, and prostate tissue. TPEF maps to human chromosome 2q33, where frequent loss of heterozygosity is seen in various human tumors, and TPEF was not expressed in most human colon and various other tumor cell lines examined by reverse transcription-PCR. Nine of 11 tumor cell lines were highly methylated in the 5' region and the first exon of the gene that demonstrated features characteristic of a CpG island. However the other two cell lines, which expressed TPEF, were hypomethylated in the 5' end of the gene. The region was also hypermethylated in 11 of 16 primary bladder tumors and in 3 of 4 primary colon tumors when compared with adjacent normal tissue. Our results suggest that potential tumor suppressor genes can be isolated from human tumors by virtue of their altered methylation patterns.

Intraocular tumor formation of RB reconstituted retinoblastoma cells.

It has been reported that replacement of a functional retinoblastoma (RB) gene in RB defective WERI-27 retinoblastoma cells results in complete loss of their tumorigenic potential in nude mice following s.c. injection. We have repeated the identical studies and found that although tumors did not develop s.c., the RB reconstituted cells, either soon after RB virus infection or after long term cultivation, consistently produced tumors when injected intraocularly. These tumor cells, when reestablished in culture, were found to retain a normal RB protein as determined by direct Western blotting and immunocytochemical staining. The tumors, however, occurred with a longer average latency period and with less frequency compared to those produced by the parental RB defective cells. Our results suggest that reintroduction of the RB gene into WERI-27 cells reduces but does not completely suppress their tumorigenic potential. Since retinoblastoma is an eye tumor it also provides further documentation that the use of an orthotopic injection site can be critical when determining the tumorigenicity of a given cell type.

The effect of myc proteins on SV40 replication in human lymphoid cells.

We have previously demonstrated that over-expression of c-myc facilitates the replication of SV40 DNA in human Burkitt lymphoma cells (BL). In this paper we investigated the ability of N-myc and L-myc to substitute for c-myc in promoting SV40 DNA replication. Vectors expressing either a chimeric N-myc/c-myc, N-myc or L-myc gene were constructed and co-transfected with a plasmid containing the SV40 origin of replication and the early genes encoding SV40 T antigens (Tag). The chimeric N-myc/c-myc and the N-myc proteins enhance SV40 replication in the human lymphoma line BJAB at levels comparable to c-myc. The stimulative effect of N-myc is also evident in the BL cell line RAMOS. However, L-myc stimulated SV40 replication only in RAMOS cells.

Nucleotide sequence of the rat Bmyc gene.

We have cloned and sequenced the rat Bmyc gene. The rat Bmyc gene contains sequences related to the central part of c-myc, namely the first intron, the second exon, and the noncoding part of the third exon. The homology drops in the 3' part of the c-myc second exon, but continues in the noncoding part of the third exon. We have sequenced the total predicted coding region of the Bmyc. The longest open reading frame in Bmyc suggests a protein of 178 amino acids, which is only 41% of the c-myc protein size. To confirm the putative open reading frame, we have produced a trpE-Bmyc protein that is detected with a pan-myc antibody. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc-family proteins.

Aberrant class switching juxtaposes c-myc with a middle repetitive element (LINE) and an IgH intron in two spontaneously arising rat immunocytomas.

Our previous studies of spontaneously arising rat immunocytomas of the Lou/Wsl strain have shown that Ig switch regions are frequently the targets for c-myc recombination. In several tumors, however, we were unable to show recombination of the c-myc with Ig switch regions. We have cloned the rearranged c-myc fragments from 2 of these tumors, IR209 and IR223, and found that the c-myc recombines with a LINE region in the IR209 and with intron 1 of the epsilon locus in the IR223. Although switch regions are not found at the breakpoints, the sequences at the breakpoints share limited homology with Ig switch recognition sequences. This suggests that the switch recombinase enzymes are able to recognize sequences in addition to the defined switch recombination sites. At the same time, both the LINE and epsilon intron 1 sequences are located within the Ig cluster, providing further evidence for the selection of c-myc activation by Ig sequences in the pathogenesis of rat immunocytoma, mouse plasmacytoma, and Burkitt's lymphoma.

Elevated expression of c-myc and N-myc produces distinct changes in nuclear fine structure and chromatin organization.

The proto-oncogenes c-myc and N-myc encode nuclear phosphoproteins with unknown function. Here, c-myc or N-myc, or hybrid constructs of the two, were transfected into fibroblastic cells (CV-1) using SV40-based high expression vectors. The cells were studied by indirect immunofluorescence microscopy and transmission electron microscopy to determine the localization of the two myc proteins within the nucleus and their influence on nuclear fine structure and chromatin organization. In c-myc transfected cells the overproduced protein product accumulated in large amorphous globules that displaced the normal chromatin and did not stain for DNA. In N-myc transfected cells condensed chromatin loops were formed. They were attached to the nuclear envelope and by traction in the latter they may have contributed to give the nucleus its irregular shape in these cells. During mitosis the chromatin loops persisted as clearly identifiable entities within the chromosomes, suggesting a rigid conformation that did not allow normal chromosome packaging. These findings suggest that the c-myc and N-myc proteins bind to different structures and may have different functions. Observations on cells transfected with hybrid constructs indicated that both the second and third exon of c-myc were required to yield a product that behaved like the c-myc protein. In contrast, domains encoded by the second exon of N-myc were sufficient to give rise to a product that morphologically behaved like the N-myc protein.

Chromosome localization and expression pattern of Lmyc and Bmyc in murine embryonal carcinoma cells.

Using Southern blot analysis of DNA from mouse-hamster somatic cell hybrids, we have mapped Lmyc and Bmyc, two members of the myc family of genes, to mouse chromosomes 4 and 2, respectively. Furthermore, we have compared the regulation of Lmyc and Bmyc expression under different growth conditions and during in vitro differentiation of the murine EC line F9 and considered the findings in relation to our previous studies on Nmyc and c-myc expression in the same line (Sejersen et al., 1987). Lmyc was down-regulated at an early stage of visceral endoderm differentiation, similarly to c-myc and Nmyc, while Bmyc was expressed at a constant low level at all stages. Lmyc, but not c-myc and Nmyc, was upregulated in terminally differentiated visceral endoderm cells. Inhibition of protein synthesis by cycloheximide for 4 h induced a 70% increase in Lmyc and 30% increase in Bmyc transcript levels, indicating that the expression of these genes is negatively regulated by a short-lived protein. Mitogenic stimulation with insulin and transferrin did not affect Lmyc and Bmyc mRNA levels. Lmyc transcripts have a half life of 30 min, whereas the Bmyc transcript is highly stable, with a half life of 6 h. The half-lives of the c-myc and Nmyc transcripts have been estimated previously as 40 and 130 min, respectively.

A new variant 15; 16 translocation in mouse plasmacytoma leads to the juxtaposition of c-myc and immunoglobulin lambda.

Mouse plasmacytomas (MPCs) induced by pristane oil, or by a combination of pristane oil and Abelson virus, carry one of two chromosomal translocations. The typical 12; 15 translocation leads to the juxtaposition of c-myc and immunoglobulin heavy-chain sequences, whereas the 6; 15 translocation links the kappa light-chain locus with the pvt-1 (plasmacytoma variant translocation) locus, located at least 75kb 3' of c-myc [Cory, S., Graham, M., Webb, E., Corcoran, L. & Adams, J. (1985). EMBO J., 4, 675-681]. Unlike the human Burkitt's lymphoma-associated translocation, the lambda/myc juxtaposed variant translocation has not been found previously in MPCs. Using unconventional MPC induction systems in which the tumor precursor cell was induced to proliferate in a secondary host, we have recently identified a 15; 16 translocation in six of the derived MPCs [Wiener, F., Silva, S., Sugiyama, H., Babonits, M. & Klein, G. (1990). Genes Chromosomes Cancer, 2, 36-43]. Chromosome 16 harbors the lambda light-chain gene. To explore whether the 15; 16 translocation represents the lambda/myc juxtaposition, we have mapped the breakpoints on chromosomes 15 and 16 by pulsed-field gel electrophoresis (PFGE). The pvt-1 region was mapped to approximately 220 kb 3' of c-myc. The breakpoint on chromosome 15 in ABPC-Ch-163-10, one of the six 15; 16 translocation-carrying MPCs, was situated approximately 80 kb 3' of c-myc and 140 kb 5' of pvt-1b, the major breakpoint cluster region of the previously analysed 6; 15 variant MPCs. The breakpoint on chromosome 16 was found to cut between the V1 and C3 regions of the lambda locus. Co-migration experiments showed that the C3 and the myc gene were juxtaposed head to tail on the 15; 16 translocation chromosome. On the reciprocal product V1 was juxtaposed to pvt-1.

The gene from the short arm of chromosome 3, at D3F15S2, frequently deleted in renal cell carcinoma, encodes acylpeptide hydrolase.

Loss or inactivation of a gene on the short arm of chromosome 3 may contribute to the genesis of renal cell carcinoma. A gene that corresponds to the most frequently lost RFLP site (D3F15S2) is expressed in a variety of human tissues, and at a particularly high level in the kidney. Its expression is markedly reduced in renal cell carcinoma. A database search showed that the gene product is closely related to or identical with acylpeptide hydrolase. The nucleotide identity between the rat acylpeptide hydrolase and the human gene at D3F15S2 is 88%, compatible with normal species differences. It is therefore likely that the human gene product is acylpeptide hydrolase. The renal cell carcinoma is then associated with a decrease of acylpeptide hydrolase activity. The gene may represent a tumor suppressor gene, whose loss contributes to the development of renal cell carcinoma. It might be speculated that it could act e.g. by affecting the activity of a small acetylated growth factor. Alternatively, its decreased expression may merely reflect the impairment of differentiation in RCC, compared to normal kidney. Loss of a linked but irrelevant gene by the 3p deletion is another possibility.

Purification and properties of the RNA polymerase of an extremely thermophilic bacterium: Thermus aquaticus T2.

The RNA polymerase of the extremely thermophilic bacterium Thermus aquaticus T 2 has been purified to homogeneity. The apparent molecular weights of the subunits of the enzyme are : 165 000, 130000, 92 000 and 44 000. The in vitro temperature optimum of enzyme activity is around 65 degrees C. The enzyme has a preference towards the homologous template and is strongly inhibited by KCI. Rifampicin inhibits the enzyme only to 50% even at very high concentrations. Heparin inhibits it completely, but only at higher molar excess than in the case of the Escherichia coli enzyme. The enzyme can form heparin-resistant complexes at elevated temperatures on the homologous template, but not on E. coli DNA.

In vitro transscription of ribosomal RNA on phage lambdarifd 18 DNA.

In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage lambdarifd 18, which carries an rRNA transcription unit from Escherichia coli. rRNA synthesis was preferential at all polymerase/DNA ratios tested, and at the optimal 0.3 weight ratio nearly 60% of the transcript was rRNA. At this ratio the principal product of transcription comigrated in acrylamide-agarose electrophoresis with authentic 30-S rRNA precursor synthesized in vivo. In the presence of rifampicin more than one equivalent of rRNA was synthesized, thus suggesting the existence of two initiation sites for rRNA on the phage DNA. Similar results were obtained on E. coli DNA. Preincubation with heparin virtually eliminated the transcription of rRNA, in sharp contrast with the results of similar experiments on bacterial DNA where rRNA genes were transcribed 4--5 times in the presence of heparin. The possible explanation of this difference between rRNA promotors on the phage and the bacterial DNA are discussed.

A new candidate region for the positional cloning of the XLP gene.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors.

By applying the 'recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5' ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.