Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.

Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines.

The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.

Delineation of the protein domains responsible for SYT, SSX, and SYT-SSX nuclear localization.

In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.

Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas.

In a previous study we reported the isolation of the human synovial sarcoma-associated t(X;18) breakpoint. As a result of this translocation, the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. As yet, little is known about the modes of action of the SYT and SSX genes and their respective (fusion) products. Here we report the isolation of the mouse homolog of SYT, its full length cDNA sequence, its chromosomal localization, and its spatio-temporal expression patterns in adult and embryonic tissues. The SYT gene was found to be well conserved during evolution and is part of a region of synteny between the human and mouse chromosomes 18. In early embryogenesis, Syt is ubiquitously expressed. In later stages, the expression becomes confined to cartilage tissues, specific neuronal cells and some epithelial derived tissues. In mature testis, expression was specifically observed in primary spermatocytes.

Normal development, growth and reproduction in cellular retinoic acid binding protein-I (CRABPI) null mutant mice.

We have generated mouse null mutants for the cellular retinoic acid (RA) binding protein type I (CRABPI), a protein whose spatio-temporal expression pattern coincides with the target tissues for RA action. Inactivation of the CRABPI gene was accomplished via homologous recombination in embryonic stem cells. Cells carrying the correctly targeted gene were injected into blastocysts and the resulting chimaeras yielded offspring heterozygous for the knockout mutation. Subsequent breeding programs resulted in normal litter sizes containing viable and fertile CRABPI deficient mice. Homozygous mice carrying the knockout mutation were studied in detail to detect possible organ and skeletal anomalies and/or abnormalities of the hematopoietic system. No overt phenotype was evident indicating that a deficiency for CRABPI does not seem to interfere with normal development, growth and reproduction.

Molecular analysis of a flagellar core protein gene of Serpulina (Treponema) hyodysenteriae.

The flaB2 gene encoding a protein located in the core of the periplasmic flagella of Serpulina hyodysenteriae was cloned and sequenced. The FlaB2 protein consists of 285 amino acids and has a calculated molecular mass of 31.1 kDa. Southern blot analysis indicated that at least one, and possibly two genes related to flaB2 are present in the genome of S. hyodysenteriae. Comparison of the amino acid sequence of FlaB2 to sequences present in data banks showed significant similarity with the core flagellins of other spirochaetes, in particular with a FlaB2 protein from Treponema phagedenis.

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.