The gene encoding the human spasmolytic protein (SML1/hSP) is in 21q 22.3, physically linked to the homologous breast cancer marker gene BCEI/pS2

The human spasmolytic protein, SML1/hSP, an inhibitor of spasmolytic activity and gastric acid secretion in the pig, has been shown to exhibit homology to the pS2 protein, an estrogen-dependent breast cancer marker. Moreover, SML1/hSP and pS2 are expressed at the same localization in the normal stomach and during healing of the gastrointestinal tract. Here we report the chromosomal localization, obtained by in situ hybridization, of the hSP gene (SML1) to chromosome 21 at 21q22.3. Using pulsed-field gel electrophoresis, we found SML1 to be within 230 kb of the BCEI/pS2 gene.     

Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21

Summary A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SMLl gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SMLl gene is also localized on chromosome 21.     

Close linkage of human chromosomal pepsinogen A genes

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.     

Oestrogen-induced pS2 protein is similar to pancreatic spasmolytic polypeptide and the kringle domain.

The phosphate complex of sulphite oxidase in the Mo(V) oxidation state was investigated by e.p.r. spectroscopy. Third-derivative spectra reveal a wealth of structural detail previously unobserved in this spectrum. Most notable is the presence of hyperfine coupling from two inequivalent I = 1/2 nuclei, which we tentatively attribute to two 31P nuclei. Unresolved hyperfine interactions from at least one exchangeable 1H nucleus are also present.     

Close physical linkage of human troponin genes: organization, sequence, and expression of the locus encoding cardiac troponin I and slow skeletal troponin T

Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.     

Primary structure of human protein pS2

We have previously reported that pS2 mRNA expressed in cultured epithelial cells derived from a hormone-dependent breast carcinoma (MCF-7 cells) is also expressed in mucosa cells of normal human stomach. This mRNA encodes a putative 84 amino-acid-long protein, which is secreted by both cell types after elimination of a signal peptide. We report here the purification of the pS2 protein, its trypsin digestion and amino-acid sequencing. The MCF-7 cell-secreted protein is 60 amino-acid-long and its sequence is in complete agreement with that deduced from the mRNA sequence. The presence of an N-terminal glutamic acid indicates that the signal peptidase releases a 24 amino-acid-long signal peptide. Analysis of tryptic peptides derived from the secreted gastric pS2 protein indicates that the signal peptide and the sequence of the first 48 amino-acids are identical to those of secreted MCF-7 pS2 protein, although the N-terminal amino-acid of the gastric protein may be cyclized as a pyroglumatic acid.     

Close physical linkage of the murine Ren-1 and Ren-2 loci

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.     

Close genetic and physical linkage between the murine haemopoietic growth factor genes GM-CSF and Multi-CSF (IL3).

The two murine haemopoietic growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF) and Multi-CSF (interleukin 3) stimulate the proliferation and differentiation of an overlapping set of haemopoietic progenitor cells and are produced coordinately following activation of T lymphocytes. Here we report the chromosomal location of the genes encoding these two factors. Initially both genes were assigned to chromosome 11 by analysis of mouse/Chinese hamster somatic cell hybrids. Genetic analysis using an interspecies (Mus musculus X Mus spretus) back-cross confirmed this assignment by demonstrating that both the GM-CSF and Multi-CSF genes are genetically linked to the SPARC gene, which had been independently assigned to sub-band B1 of chromosome 11. Analysis of physical distances by pulsed field gel electrophoresis demonstrated further that the two CSF genes lie within 230 kb of each other. However examination of the subchromosomal region containing all three loci by pulsed field gel analysis showed that SPARC is at least 400-500 kb distant from the region containing the two CSF genes.     

Close linkage of genes encoding glutamine synthetases I and II in Frankia alni CpI1.

Frankia alni CpI1 has two glutamine synthetases (GSs), GSI and GSII. The GSI gene (glnA) was isolated from a cosmid library of F. alni CpI1 DNA by heterologous probing with glnA from Streptomyces coelicolor. The glnA gene was shown to be located upstream of the GSII gene (glnII) by DNA-DNA hybridization. The nucleotide sequences of the 1,422-bp CpI1 glnA gene and of the 449-bp intervening region between glnA and glnII were determined, and the glnA amino acid sequence was deduced. In common with GSIs from other organisms, CpI1 GSI contains five conserved regions near the active site and a conserved tyrosine at the adenylylation site. F. alni CpI1 glnA complemented the glutamine growth requirement of the Escherichia coli glnA deletion strain YMC11 but only when expressed from an E. coli lac promoter. While the functional significance of maintaining two GSs adjacent to one another remains unclear, this arrangement in F. alni provides support for the recently proposed origin of GSI and GSII as resulting from a gene duplication early in the evolution of life.     

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.     

Effects of tamoxifen and 4-hydroxytamoxifen on the pNR-1 and pNR-2 estrogen-regulated RNAs in human breast cancer cells

The estrogenic and antiestrogenic activities of tamoxifen and 4-hydroxytamoxifen have been measured on the expression of two estrogen-regulated RNAs (pNR-1 and pNR-2) in the MCF7 human breast cancer cell line cultured in phenol red-free medium. The two antiestrogens increased the level of the pNR-1 RNA to about 80% of the estradiol-induced level, and the induction by estradiol was not significantly antagonized by either antiestrogen. In contrast, the pNR-2 mRNA was only increased to about 10% of the estradiol-induced level, and its induction by estradiol was antagonized by both tamoxifen and 4-hydroxytamoxifen. Thus, the two RNAs respond in dramatically different ways to these antiestrogens. 4-Hydroxytamoxifen and estradiol have similar affinities for the estrogen receptor; however, the induction of both RNAs by 4-hydroxytamoxifen required a 10-fold higher concentration than estradiol for maximum agonist activity, and a 500-fold molar excess was required to antagonize the induction by estradiol. Tamoxifen has a 20-100-fold lower affinity than estradiol for the estrogen receptor. A 200-fold higher concentration was required for maximum agonist activity and a 10,000-fold molar excess to antagonize the induction by estradiol. These results emphasize the complexity of antiestrogen action in human breast cancer cells.     

Expression of the oestrogen responsive protein pS2 in human breast cancer

The trefoil peptide pS2 was discovered in a breast cancer cell line as a result of its oestrogen responsive character. The expression of pS2 in breast tumours in vivo is also likely to be an oestrogenic effect and as such, the presence of pS2 in oestrogen receptor positive breast cancer is evidence of an intact oestrogen response pathway and an indicator of putative hormone responsiveness. Consistent with this, clinical studies of breast cancer have revealed a correlation between pS2 expression and favourable tumour characteristics as well as response to endocrine therapy.     

Estrogen-induced genes,WISP-2 and pS2, respond divergently to protein kinase pathway

Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.     

Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.)

The maize genomic sequence and cDNA encoding a calcium/calmodulin-dependent protein kinase homolog were isolated and identified. The deduced peptide (MCK2) from this cDNA shared high amino acid identity (91.2%) with maize MCK1. These two genes were physically mapped onto chromosomes by fluorescence in situ hybridization using the first introns of the genes as gene-specific probes. While the MCK1 gene was assigned to a locus on the long arm of chromosome 9, the MCK2 gene was localized to a locus on the long arm of chromosome 1. Both of these genes were expressed in roots, leaves, stems and flowers, and the expression patterns of MCK were verified by RNA in situ hybridization. These results indicated that MCK expression is temporally and spatially regulated during maize growth and development.