Identification and genomic organization of a gene coding for a new member of the cell adhesion molecule family mapping to Xq25.

The gene coding for a new member of the Immunoglobulin (Ig)-like domain-containing molecule superfamily has been identified and mapped to the human Xq25 chromosomal band. It contains 12 Ig-like domains in two clusters of 5 and 7 motifs, respectively, separated by a linker segment, followed by a transmembrane and a cytoplasmic region. The gene is conserved in mammals and is expressed in muscle, heart, brain, testis, and pancreas with transcripts of different length, suggesting that it is subjected to alternative processing. The transcript is assembled from 19 exons which are distributed along approx. 20kb; each Ig-like domain is contained in distinct exons which constitute the unit of repeated genomic duplications. Elucidation of the IGDC1 genomic structure will allow the investigation of the basis of its alternative transcription and of its possible involvement in diseases mapped to the Xq25 interval.

Identification of a new member (ZNF183) of the Ring finger gene family in Xq24-25.

Four genes were mapped to the Xq24-25 region by searching the EST and the non-redundant database with short tracts of genomic sequences. These were random STSs present in the STS database or sequences derived from CpG islands (EagI-based STSs). One of the four matches corresponded to the full length transcript from the intronless glutamate dehydrogenase gene. The second was the human homolog of the bovine NADH ubiquinone oxidoreductase MWFE subunit gene (GDB symbol: NDUFA1). The other two, ZNF183 and ITBA4, were novel genes whose function cannot directly be inferred from their sequence analysis. However, a known motif, the C3HC4 Ring finger domain, shared by various tumor suppressors, DNA repair genes and cytokine receptor-associated molecules, is present at the C terminus of the ubiquitously expressed ZNF183 gene. ITBA4 is expressed at various levels in different tissues and is alternatively processed in brain. Similarity search did not detect any significant match in databases. These results, together with others previously reported by our laboratory, suggest that comparison of genomic and transcribed sequences which are continuously accumulating in databases, can provide 'virtual' mapping of a substantial number of ESTs to the specific genomic region which the STSs have been derived from.

Partial regression, yet incomplete eradication of mammary tumors in transgenic mice by retrovirally mediated HSVtk transfer 'in vivo'.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

Computer gene mapping by Eagl-based STSs.

The rapid pace at which the human genome project has proceeded has greatly benefited from two classes of short sequence tags, genomic (STS) and transcribed (EST), which are listed in two separate databases. Usually, STSs are random genomic sequences derived only for mapping purposes, while ESTs represent transcribed sequences that have to be mapped one by one. Here, we propose a way of establishing links between these two sets of sequences, allowing the automatic mapping of EST sequences by simple comparison with relatively nonrandom STSs. We suggest that EagI-based STSs derived by selected genomic portions organized in YAC contigs can automatically finely map a relevant portion of the ESTs, partially bridging the gap between the two sets of sequences and saving a great amount of time in mapping efforts. To test this principle, we have selected 330 high-quality STSs derived from the Xq24-qter region and used them for transcript searches by comparing them to the EST as well as to the nonredundant database. This search detected four known genes and two additional EST clones. In contrast, when the same databases were searched with a set of 53 sequences derived from the same chromosomal region around EagI sites, 7 known genes and 6 additional ESTs were found. These findings, together with data obtained from simulation analysis on long sequences in the same chromosomal region, suggest that EagI-based STSs can partially bridge the gap between STSs and ESTs.

The ZNF75 zinc finger gene subfamily: isolation and mapping of the four members in humans and great apes.

We have previously reported (Villa et al. (1993), Genomics 18: 223) the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain an ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and orangutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existence of at least two genes in cows and horses, suggests a relatively high conservation of this gene family.

Characterization and fine localization of two new genes in Xq28 using the genomic sequence/EST database screening approach.

Two new genes were identified and mapped by searching the EST databases with genomic sequences obtained from putative CpG islands of the rodent-human hybrid X3000. Previous mapping of these CpG islands in the proximity of the host cell factor (HCFC1) and GdX genes automatically localized these two new genes to Xq28 in the interval between the L1 cell adhesion molecule (L1CAM) and the glucose-6-phosphate dehydrogenase (G6PD) loci. Both genes are relatively short, contain an ORF of 261 and 105 amino acids, respectively, and are ubiquitously expressed. Combining sequencing of selected CpG islands, derived from hybrids containing small portions of the human genome, with an EST database search is an easy method of identifying and mapping new genes to specific regions of the genome.

The complete sequence of the host cell factor 1 (HCFC1) gene and its promoter: a role for YY1 transcription factor in the regulation of its expression.

We report here the complete sequence of the Host Cell Factor (HCFC1) gene, including two kilobases of the 5'-flanking region and 5.9 kb of the first intron. The upstream and 5'-untranslated regions contain several putative transcriptional factor binding sites and a 17-nt-long repeated element (SiSa element) present in six regularly spaced copies, of which five are perfectly identical, while the sixth has a transition substitution (CT for TC) at nucleotides 13 and 14. Four copies are contained in the flanking region, the fifth is at the beginning of the mRNA (position +9), and the sixth is at position 195 of the mRNA. This 17-bp element contains at its 5' side an octamer sequence known to bind the Yin/Yang 1 (YY1) transcription factor; another YY1 binding octamer is present at the end of the first intron. The promoter also contains several Sp1 binding sites, some of which are located very close to SiSa elements. We demonstrate that YY1 binds to the 5' half of the SiSa element, whose 3' region binds in gel shift experiments an additional, as yet unidentified nuclear factor. Therefore the YY1 binding site in HCFC1 overlaps the site of a second factor, as has been described in several YY1-site-containing promoters. This suggests that HCFC1 expression might be regulated by the reciprocal interaction of several transcription factors.

The chromosome localization and the HCF repeats of the human host cell factor gene (HCFC1) are conserved in the mouse homologue.

The gene encoding the human host cell factor (HCFC1) has recently been cloned and mapped to Xq28. HCFC1 codes for a family of related polypeptides that apparently arise from posttranslational processing. Six extremely conserved 19-amino-acid (aa)long motifs, unique to HCFC1 and located in the middle of the protein, could play a role in this processing or could be instrumental to the physiological role of the protein. Aternatively, these repeats could have arisen from recent duplications and may not have any specific function. To resolve this issue, we cloned the homologous region from the mouse HCFC1 gene and demonstrated that the 19-aa motifs are extremely conserved in sequence, number, and genomic organization, while the "linker" region between the third and fourth repeat is not. This suggests an important function for these repeats. In addition, by RT-PCR analysis of human RNA and comparison to the human genomic sequence, an alternative transcript including a 44-aa in-frame insertion, deriving from the 3' end of intron 18, was found. The significance of this alternative transcript is unknown, since it was not detectable in the mouse. The mouse HCFC1 gene maps to a region syntenic to Xq28, and, as in human, is in close proximity to the Renin-binding protein gene, in a 100-kb region also including the Licam and Vasopressin receptor type 2 genes.

The human genes encoding renin-binding protein and host cell factor are closely linked in Xq28 and transcribed in the same direction.

The Xq28 chromosomal band represents a C+G-rich region onto which several genes have been mapped. In most cases, the exact relationship between the mapped genes has not yet been established, and neither the regulatory nor the spacer regions between the various transcription units have been defined. In the region around the L1CAM gene (encoding L1 cell adhesion molecule), the transcription units appear, from preliminary analyses, to be quite compact. By sequencing the region at the 3' end of the recently found host cell factor 1-encoding gene (HCFC1), we report that the renin-binding protein-encoding gene (RBP) major transcription start point lies 2763 bp downstream from the 3' end of HCFC1 and that both are transcribed in the same direction from the telomere to the centromere.

Genomic organization of the human VP16 accessory protein, a housekeeping gene (HCFC1) mapping to Xq28.

The region between DXS52 and Factor VIII gene in the human Xq28 chromosomal band contains a G+C-rich isochore to which many genes have been mapped. We report here the isolation and characterization of a transcript mapping about 50 kb telomeric from the vasopressin type 2 receptor gene in a 180-kb YACs/cosmid contig containing the L1CAM gene at its centromeric end. The determined transcribed sequence from a human fetal brain library is identical to that of the recently identified accessory protein HCFC1 (host cell factor, also called C1) that activates herpes simplex virus VP16 (alpha TIF) transactivator protein for association with the octamer motif-binding protein Oct-1 (Cell 74: 115, 1993). The gene is expressed in a ubiquitous pattern and a larger transcript of approximately 10 kb is present in all the tissues tested, while an alternatively spliced RNA of approximately 8.0 kb is present in muscle and heart tissues. Genomic sequencing allowed us to determine that the sequenced transcript is assembled from 26 exons spread over a relatively small genomic region of approximately 24 kb. This alllowed us to determine that a previously reported cDNA clone arises from the splicing out of an internal portion of exon 8 which does not change the reading frame. All together these results raise the possibility that alternative mRNA processing could partly contribute to the diversity of the polypeptide HCFC1 family in a subset of tissues.

The exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280.

We have determined the exon-intron organization of the human X-linked gene (FLN1) encoding actin-binding protein 280 (filamin), a ubiquitous protein that plays an important role in the mechanochemical activities of cells through its association with actin filaments and membrane components. The gene is composed of 47 exons spanning approximately 26 kb. The first and part of the second exon are untranslated. The actin-binding domain at the N-terminus is encoded by exons 2 to 5. The 96-amino-acid repeats corresponding to the elongated rod backbone of the protein are encoded by the remaining 42 exons: size, location, and boundaries of the exons cannot be easily correlated with the repeated structure, while sequences interrupting the repeats (the two hinge segments preceding repeats 16 and 24 and the 8-amino-acid (aa) segment interrupting the 15th repeat) were encoded by separate exons, suggesting that they may be recent additions to the X-linked protein. The 8-aa segment is encoded by exon 29, which is alternatively spliced.