Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time.

The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.

Spleen tyrosine kinase (Syk) deficiency in childhood pro-B cell acute lymphoblastic leukemia.

The cytoplasmic spleen tyrosine kinase (SYK) is a key regulator of signal transduction events, apoptosis and orderly cell cycle progression in B-lineage lymphoid cells. Although SYK has not been linked to a human disease, defective expression of the closely related T-cell tyrosine kinase ZAP-70 has been associated with severe combined immunodeficiency. Childhood CD19(+)CD10(-) pro-B cell acute lymphoblastic leukemia (ALL) is thought to originate from B-cell precursors with a maturational arrest at the pro-B cell stage and it is associated with poor prognosis. Since lethally irradiated mice reconstituted with SYK-deficient fetal liver-derived lymphohematopoietic progenitor cells show a block in B-cell ontogeny at the pro-B to pre-B cell transition, we examined the SYK expression profiles of primary leukemic cells from children with pro-B cell ALL. Here we report that leukemic cells from pediatric CD19(+)CD10(-) pro-B cell ALL patients (but not leukemic cells from patients with CD19(+)CD10(+) common pre-pre-B cell ALL) have markedly reduced SYK activity. Sequencing of the reverse transcriptase-polymerase chain reaction (RT-PCR) products of the Syk mRNA in these pro-B leukemia cells revealed profoundly aberrant coding sequences with deletions or insertions. These mRNA species encode abnormal SYK proteins with a missing or truncated catalytic kinase domain. In contrast to pro-B leukemia cells, pre-pre-B leukemia cells from children with CD19(+)CD10(+) common B-lineage ALL and EBV-transformed B-cell lines from healthy volunteers expressed wild-type Syk coding sequences. Examination of the genomic structure of the Syk gene by inter-exonic PCR and genomic cloning demonstrated that the deletions and insertions in the abnormal mRNA species of pro-B leukemia cells are caused by aberrant splicing resulting in either mis-splicing, exon skipping or inclusion of alternative exons, consistent with an abnormal posttranscriptional regulation of alternative splicing of Syk pre-mRNA. Our findings link for the first time specific molecular defects involving the Syk gene to an immunophenotypically distinct category of childhood ALL. To our knowledge, this is the first discovery of a specific tyrosine kinase deficiency in a human hematologic malignancy.

Expression of aberrantly spliced oncogenic ikaros isoforms in childhood acute lymphoblastic leukemia.

PURPOSE: We sought to determine if molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in children. PATIENTS AND METHODS: We studied Ikaros gene expression in normal human bone marrow, normal thymocytes, normal fetal liver-derived immature lymphocyte precursor cell lines, eight different ALL cell lines, and leukemic cells from 69 children with ALL (T-lineage ALL, n = 18; B-lineage ALL, n = 51). Expression of Ikaros protein and its subcellular localization were examined by immunoblotting and confocal laser-scanning microscopy, respectively. Polymerase chain reaction (PCR) and nucleotide sequencing were used to identify the specific Ikaros isoforms expressed in these cells. Genomic sequencing of splice junction regions of the Ikaros gene was performed in search for mutations. RESULTS: In each of the ALL cases, we found high-level expression of a non-DNA-binding or aberrant DNA-binding isoform of Ikaros with abnormal subcellular compartmentalization patterns. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal subcellular localization were found in normal bone marrow cells and thymus-derived or fetal liver-derived normal lymphocyte precursors. In leukemic cells expressing the aberrant Ikaros coding sequences with the 30-base-pair deletion, genomic sequence analysis of the intron-exon junctions between exons 6 and 7 yielded the wild-type sequence. We identified a single nucleotide polymorphism (SNP) affecting the third base of the triplet codon for a proline (CCC or CCA) in the highly conserved bipartite activation region (viz, A or C at position 1002 numbering from the translation start site of Ik-1) within our Ikaros clones. Bi-allelic expression of truncated and/or non-DNA-binding isoforms along with wild-type isoforms was observed in leukemic cells, which implicates trans-acting factor(s) affecting splice site recognition. CONCLUSION: Our findings link specific molecular defects involving the Ikaros gene to childhood ALL. Posttranscriptional regulation of alternative splicing of Ikaros RNA seems to be defective in leukemic lymphocyte precursors from most children with ALL. Consequently, leukemic cells from ALL patients, in contrast to normal lymphocyte precursors, express high levels of non-DNA-binding Ikaros isoforms that are reminiscent of the non-DNA-binding Ikaros isoforms that lead to lymphoblastic leukemia in mice.

Production of inhibitor of insulin-receptor tyrosine kinase in fibroblasts from patient with insulin resistance and NIDDM.

Although non-insulin-dependent diabetes mellitus (NIDDM) is associated with defects in insulin action, the molecular basis of this resistance is unknown. We studied fibroblasts from a markedly insulin-resistant patient with NIDDM but without acanthosis nigricans. Her fibroblasts were resistant to insulin when alpha-aminoisobutyric acid uptake was measured. Fibroblasts from this patient demonstrated normal insulin-receptor content as measured by both insulin-receptor radioimmunoassay and by Scatchard analysis. However, when compared with nondiabetic control subjects, insulin-receptor kinase assays of wheat-germ-purified receptors prepared from her fibroblasts showed very low basal and no insulin-stimulated tyrosine kinase activity. The insulin receptor was then removed from the wheat-germ fraction by monoclonal antibody affinity chromatography. This insulin-receptor-deficient fraction inhibited both basal and insulin-stimulated tyrosine kinase activity of highly purified insulin receptors. When the specificity of this inhibition was tested, less inhibition was seen with insulinlike growth factor I-receptor tyrosine kinase, and even less inhibition was seen with the proto-oncogene p60c-src tyrosine kinase. Thus, these studies indicate that fibroblasts from an insulin-resistant patient with NIDDM produce a relatively specific glycoprotein inhibitor of insulin-receptor tyrosine kinase. Therefore, these studies raise the possibility that this inhibitor may play an important role in the insulin resistance seen in this patient.

Many protein products from a few loci: assignment of human salivary proline-rich proteins to specific loci.

Earlier studies of protein polymorphisms led to the description of 13 linked loci thought to encode the human salivary proline-rich proteins (PRPs). However, more recent studies at the DNA level have shown that there are only six genes which encode PRPs. The present study was undertaken in order to reconcile these observations. Nucleotide and decoded amino acid sequences from each of the six genes were compared with the available protein sequence data for PRPs. This analysis allowed assignment of the PmF, PmS and Pe proteins to the PRB1 locus, the G1 protein to the PRB3 locus, the Po protein to the PRB4 locus, the Ps protein to the PRB2 locus, and the CON1 and CON2 proteins to the PRB4 locus. Correlations between insertion/deletion RFLPs and PRP protein phenotypes were observed for the PmF, PmS, Gl and CON2 proteins. Our overall analysis indicates that in many instances several proteins previously considered to be the products of separate loci are actually proteolytic cleavage products of a large precursor specified by one or other of the six genes identified at the DNA level. Our analysis also demonstrates that some of the "null" alleles proposed to occur at 11 of the 13 loci in the earlier genetic studies, are actually productive alleles having alterations at proteolytic cleavage sites within the relevant precursor protein. The absence of cleavage leads to the persistence of longer precursor peptides not resolved electrophoretically, concurrently with an absence of the smaller PRPs seen when cleavage occurs.

Rational drug design of multifunctional phosphoramidate substituted nucleoside analogs.

This review focuses on our approach to the study of the effect of a series of phosphoramidate substituted nucleoside analogs on model systems for cancer, HIV and fertility. This approach allowed the development of compound WHI-07, an arylphosphoramidate derivative of zidavudine. This compound is a multifunctional agent showing potent activity in the above mentioned model systems. Our rational drug design provided such a powerful derivative with all the necessary characteristic of a drug candidate. Importantly, we have experimental evidence that each of the groups associated with the molecular frame of WHI-07 imparts the multifunctional ability for this agent. In addition, we have also suggested a possible biological pathway for WHI-07 including various products with their therapeutic targets that are formed during the course of its metabolism inside the cell. We also propose which individual moieties in the structure of WHI-07 are responsible for the biological activity from the formation of these metabolites. A detailed structure-activity relationship is presented in the review in connection with various structural modifications of the agent. Application of this active agent in animal models shows the potential usefulness of this agent as a drug candidate. We further plan to utilize gene-chip technology to identify new targets and modes of action using microarrays to measure expression changes in thousands of gene products. In conclusion, we have demonstrated the power of multifunctional drug design to discover drugs to combat various diseases. We believe this is the future direction of the drug discovery process.

Identification of the human insulin negative regulatory element as a negative glucocorticoid response element.

Insulin gene transcription in adults is restricted to pancreatic beta cells. Studies with both transgenic mice and islet cell lines have demonstrated that beta cell specific expression is conferred by the 5' flanking region of the insulin gene. Transfection analysis has shown that cell specific expression involved an interaction between both positive and negative promoter cis elements. An upstream region (between -258 and -279) of the human insulin promoter served as a site of negative regulation. Transfection analysis in the pancreatic cell line HIT T-15 M 2.2.2 revealed that a DNA fragment containing this region causes a 45% reduction in promoter activity when linked to the native insulin promoter and a 72% reduction when linked to a heterologous tk promoter. Electrophoretic mobility shift analysis of this negative regulatory region (NRE) reveals a complex pattern of binding, wherein two major and several minor complexes are observed. Competition experiments demonstrated that formation of the fastest mobility complex is completely inhibited with excess cold glucocorticoid responsive element (GRE) consensus oligonucleotide. Purified glucocorticoid receptor binding domain (T7X556) demonstrated binding to the NRE oligonucleotide. Functional studies showed that dexamethasone treatment of HIT T-15 M 2.2.2 cells containing an NRE-tk CAT plasmid decreased CAT gene expression by 48%. Analysis of the NRE revealed 73% homology with the negative GRE consensus sequence. These data show that the human insulin NRE is a negative glucocorticoid response element.

Growth factor receptor regulation in the Minn-1 leprechaun: defects in both insulin receptor and epidermal growth factor receptor gene expression.

Leprechaunism is a disorder characterized by intrauterine growth retardation, distinctive dysmorphology, and extreme insulin resistance due to structural abnormalities of the insulin receptor (IR). In addition to the IR, it has been suggested that abnormalities of the other growth factor receptors may occur in this syndrome. Using fibroblasts from the Minn-1 leprechaun, we have now investigated the expression of three different growth factor receptor genes: the IR, the insulin-like growth factor-I receptor (IGF-IR), and the epidermal growth factor receptor (EGFR). In agreement with previous studies, we found decreased insulin binding to fibroblasts from the Minn-1 leprechaun. In these cells, the IR transcription rate was not decreased, and sequence analysis of the IR promoter region of the patient showed no abnormalities. Both single-stranded conformational polymorphism analysis (SSCP) and DNA sequencing confirmed a previously reported nonsense mutation in one of the patient's two IR alleles at exon 14. mRNA levels for the IR were markedly decreased, suggesting that IR mRNA turnover was enhanced. We then studied the expression of the closely related IGF-IR Ligand binding, mRNA content, and transcription rate were all normal. In contrast to the IGF-IR, when the EGFR was studied, ligand binding and mRNA content were markedly decreased. These studies therefore raise the possibility that the phenotypic expression of leprechaunism results from defects in the expression of both the IR and the EGFR.

Needle localization of nonpalpable breast masses.

A series of 146 women underwent 150 preoperative localizations of mammographically suspicious but nonpalpable breast lesions. The lesions were localized using the hook-wire method of Frank in 133 of these patients. Carcinoma was discovered in 24 (16%) of the women; 18 (75%) of these women had invasive and six women (25%) had noninvasive carcinomas. Sixty-seven patients demonstrated calcification, and of these, 16 patients (24%) turned out to have malignancies. Eighty percent of the cancers were less than 1 cm in diameter, and 38% met the criteria of minimal carcinoma as described by Gallagher and Martin in 1969. Fourteen percent of the patients with carcinoma had lymph node metastases. We conclude that this is a safe, rapid, and accurate method for localizing small, potentially highly curable breast cancers with minimal sacrifice of breast tissue.

The human insulin gene contains multiple transcriptional elements that respond to glucocorticoids.

Glucocorticoids inhibit insulin expression in cultured pancreatic islet cells. In this study, we provide evidence that transcriptional downregulation of insulin gene expression by glucocorticoids is the result of synergistic interaction between various elements of the insulin promoter. Similar synergistic effects on insulin gene transcription were previously reported for other key insulin regulators, cyclic adenosine monophosphate (cAMP) and glucose. Transfection of CAT constructs containing different segments of the insulin promoter into the pancreatic cell line, HIT T-15 2.2.2, demonstrated that dexamethasone decreased CAT activity in all constructs tested. However, differences were found in the relative sensitivities of the various constructs. Glucocorticoid inhibition of expression from plasmids containing A elements may result from decreased expression of the pancreatic homeodomain protein STF-1. However, a different mechanism must be invoked for insulin promoter constructs lacking A sites, which nevertheless still demonstrated negative regulation. Glucocorticoid-induced inhibition of one of these regions (-882 to -342) was seen to require pancreas-specific factors, because inhibition was observed in HIT T-15 2.2.2 cells but not in the nonpancreatic COS-1 cells. We conclude, therefore, that the human insulin gene contains multiple transcriptional elements that respond to glucocorticoids, some of which require beta cell-specific factors.

Human parotid size polymorphism (Ps): characterization of two allelic products, Ps 1 and 2, by limited proteolysis.

The allelically determined human salivary proteins, Ps 1 and 2, were purified on sodium dodecyl sulfate gels, eluted, and compared by limited proteolysis with Streptomyces griseus protease VI, Bacillus subtilis protease VII, and Staphylococcus aureus protease V8. Prior dansylation of the Ps isoproteins facilitated visualization of the peptides. Digestion patterns indicate considerable homology between the Ps isoproteins and support the conclusion [Azen, A. E., and Denniston, C. (1980). Biochem. Genet. 18:483] that there is an actual molecular weight difference between them. Further, the results suggest that this difference owes to an extension of the Ps 2 chain at one of its ends.

Glioblastoma multiforme with extraneural metastases in the absence of previous surgery.

A 63-year-old man was found to have an intracerebral glioblastoma multiforme and preoperative roentgenographic evidence of a mass in the middle lobe of the right lung. Because of the rarity of extraneural metastases from glioblastoma, especially in the absence of prior surgery, the lesions were considered to be separate neoplasms until death. The histologic appearance of the lung tumor obtained at autopsy was identical to the cerebral tumor. Additional metastases were found to bronchial lymph nodes and a lumbar vertebra. This case demonstrates that a glioblastoma can spontaneously metastasize extraneurally. Invasion of the glioblastoma into the lumen of a blood vessel was demonstrated within the primary tumor. Embolization of cells to the lung and beyond is the suspected mode of spread.

Human salivary proline-rich protein genes on chromosome 12.

A DNA probe (PRP1) for the proline-rich protein (PRP) genes was used to analyze the segregation of human PRP genes in human X mouse somatic cell hybrids. Endonuclease restriction analysis of 22 independent hybrid clones segregating human chromosomes demonstrated that PRP genes segregate with human chromosome 12 only and were therefore assigned to that chromosome. The PRP1 probe should prove useful for further mapping studies of human chromosome 12.