Cloning and characterization of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28-32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissue-restricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.

Molecular cloning of a gene on chromosome 19q12 coding for a novel intracellular protein: analysis of expression in human and mouse tissues and in human tumor cells, particularly Reed-Sternberg cells in Hodgkin disease.

A novel protein, named NNX3, was molecularly characterized by cloning its cDNA, and its gene was mapped to chromosome 19q12. The equivalent mouse cDNA and gene were also cloned to allow us to analyze expression in murine in addition to human cells and tissues. Human and mouse NNX3 genes are composed of nine exons coding for proteins that are unrelated to any known protein. Signal peptides and hydrophobic domains are absent, corroborating their localization in the cytoplasm in transfected Cos cells. In Western blotting and immunoprecipitation, human NNX3 appeared as a doublet of Mr 64K-66K, while mouse NNX3 was a 70-kDa protein, both apparently much larger than the predicted 50 kDa, due in part to a stretch of 16-18 acidic residues hinging two nearly equally sized domains. In addition, phosphorylation of serine residues was demonstrated. Putative nuclear targeting signals were predicted, but NNX3 protein and two truncated versions remained localized in the cytoplasm of transfected Cos cells. NNX3 was expressed in embryonic and adult mouse tissues, particularly in brain, muscle, and lung. The expression of human NNX3 was most notable in human skeletal muscle and in ganglion cells and was also evident in human tumors and derived cell lines. This was confirmed by entries appearing in the GenBank EST database during the later phase of this study, representing partial NNX3 cDNA isolated from diverse neoplastic and developing tissues. Surprisingly, NNX3 was immunochemically detected in Reed-Sternberg cells of Hodgkin disease, in parallel with restin, a cytoplasmic protein we previously characterized (J. Delabie et al., 1993, Leuk. Lymphoma 12, 21-26). The cloning and comprehensive molecular analysis of NNX3 as presented will form the basis for elucidating its function and, conversely, will constitute a marker for Reed-Sternberg cells in Hodgkin disease.

Homozygous deletions of human chromosome 3p in lung tumors.

Cytogenetic and loss of heterozygosity (LOH) studies have demonstrated that deletions of chromosome 3p occur at a high frequency in all forms of lung cancer. To clarify the role of 3p in lung tumorigenesis and to more precisely identify targets for positional cloning efforts, we have performed 3p deletion analyses (microsatellite and fluorescence in situ hybridization) in a series of lung cancer cell lines and uncultured tumor samples. Importantly, we identified homozygous deletions in four uncultured tumors and one cell line. Homozygous deletions were found in three squamous tumors within a region of 3p21 which had previously been described only in cell lines, a 1-2-megabase homozygous deletion in a small cell tumor at 3p12, and a 3p14.2 homozygous deletion in a non-small cell lung carcinoma cell line. The detection of homozygous deletions affecting these multiple regions in uncultured tumor cells substantiates the belief (previously based on deletions found only in tumor cell lines) that these sites contain important tumor suppressor genes. Along with previously reported homozygous deletions in a distal portion of 3p21.3, we now have evidence for four separate regions of 3p which undergo homozygous deletions in either uncultured lung tumors or cell lines.

Molecular cloning and characterization of the human interleukin-11 receptor alpha-chain gene, IL11RA, located on chromosome 9p13.

The human gene coding for the interleukin-11 receptor (IL11RA) was cloned and its structure analyzed. The gene is composed of 13 exons comprising nearly 10 kb of DNA that was completely sequenced. The intron-exon boundaries were determined based on the mouse Etl2 and interleukin-11 receptor cDNAs that were recently cloned. The protein sequence predicted by the human gene was over 83% identical with its murine counterpart, with very strict conservation of functionally important domains and signatures. Fluorescence in situ hybridization showed the gene to be located on human chromosome 9p13, syntenic with the mouse etl2 gene on chromosome 4. The coding exons of the Interleukin-11 gene were sequenced in a patient with the cartilage-hair hypoplasia syndrome, which has been linked to a gene on chromosome 9, but no functional mutations were detected.

Trisomy 21 in solitary fibrous tumor.

We found trisomy 21 as the sole chromosome abnormality in a solitary fibrous tumor arising at a nonpleural site. No cytogenetic investigation of solitary fibrous tumors has previously been reported.

Cloning, characterization, and chromosomal localization to 4p16 of the human gene (LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein and structural comparison with the murine gene coding for the 44-kDa heparin-binding protein.

We report the molecular cloning of the human gene (symbol LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein (A2MRAP), as well as the gene coding for the 44-kDa heparin-binding protein (HBP-44), its murine counterpart. For both, genomic cosmid clones were isolated, and for the human gene a bacteriophage P1 clone containing the entire A2MRAP gene was also retrieved. The genes were characterized after subcloning: in both species, the known coding part of the cDNA is encoded by eight exons, and the position of the boundaries of the exons was conserved. The human LRPAP1 locus was assigned to chromosome 4 by PCR of human-hamster hybrid cell lines and by fluorescence in situ hybridization to band 4p16.3. This maps closely to the variable constitutional deletions of the short arm of chromosome 4, observed cytogenetically in patients with the Wolf-Hirschhorn syndrome. Metaphase spreads of two such patients were analyzed by fluorescence in situ hybridization with an LRPAP1 genomic probe. The first patient, with karyotype 46,XY,del4(p14-p16.1), had retained both copies of the LRPAP1 gene. In contrast, the other patient, with karyotype 46,XY,del4(p15.3-pter), displayed no signal for LRPAP1 on the deleted chromosome.

Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein.

The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR.

Mild phenotype and normal gonadal function in females with 4p trisomy due to unbalanced t(X;4)(p22.1;p14).

In this report we describe the mild phenotypic manifestations and normal gonadal function in a 50-year-old mother and her 17-year-old daughter with 4p trisomy resulting from an unbalanced t(X;4)(p22.1;p14) mat. Late replication of the der(X) was demonstrated by BrdU incorporation with spreading of the late replication to the 4p portion as an explanation of the reduced effect of the trisomy 4p/monosomy Xp on the physical and secondary sexual development in both patients.

Molecular cloning of the mouse gene coding for alpha 2-macroglobulin and targeting of the gene in embryonic stem cells.

We have cloned the mouse gene coding for alpha 2-macroglobulin in overlapping lambda clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5' flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phase A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

Identification of four genes coding for isoforms of murinoglobulin, the monomeric mouse alpha 2-macroglobulin: characterization of the exons coding for the bait region.

Murinoglobulins are the single chain members of the alpha 2-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each alpha-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse alpha 2-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents a genetic repertoire of the alpha 2-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.

Localization of the gene (RSN) coding for restin, a marker for Reed-Sternberg cells in Hodgkin's disease, to human chromosome band 12q24.3 and YAC cloning of the locus.

A novel 160-kDa intermediate filament associated protein, named restin (Reed-Sternberg intermediate filament associated protein), is specifically expressed in the malignant cells of Hodgkin's disease and anaplastic large cell lymphoma (Ki-1 lymphoma). The combination of chromosomal R-banding and fluorescence in situ hybridization (FISH) with the use of two fluorescent dyes, fluorescein isothiocyanate and propidium iodide, allowed simultaneous detection of the hybridized DNA sequence and chromosomal R-banding. By this technique, the gene coding for restin (RSN) was assigned to chromosome region 12q24.31-->q24.33, while localization of the alpha-2-macroglobulin receptor (A2MR) was refined to 12q13.1-->q13.3. To further analyze the restin gene, a 500-kb YAC clone containing the gene was isolated and analyzed. A restriction map of this area is presented.

Detection of amplified sequences at 5q11-->q13 in a homogenously staining region found by fluorescent in situ hybridization in a case of B-cell non-Hodgkin's lymphoma.

Gene amplification is one of the molecular mechanisms possibly involved in the initiation of tumorigenesis. Homogenously staining regions and double minutes have been shown to contain amplified DNA sequences. We report here a case of B-cell non-Hodgkin's lymphoma (NHL) carrying an abnormal chromosome 5 with an hsr-like region at 5q11-->q13. Chromosome "painting" with a chromosome 5-specific library (pBS5) showed that the amplified material was indeed derived from chromosome 5. Further identification of the amplified DNA sequences was performed with a set of YAC probes localized to 5q12-->q14. The amplified region contained genetic loci defined by D5S6, D5S125, D5S112, and D5S39. A three-fold level of DNA amplification was visualized by Southern blotting using a DNA probe from the amplified region. The present results suggest that unknown gene(s) at 5q11-->q13 might be involved in the process of lymphomagenesis when amplification occurs.

Molecular cytogenetic analysis of a familial pericentric inversion of chromosome 12.

We describe the application of multi-color fluorescence in situ hybridization (FISH) in the characterization of a familial pericentric inversion. Using chromosome 12 short- and long-arm specific DNA probes, fast and reliable discrimination between normal and inversion chromosome 12 or recombinant inversion chromosome 12 was possible. FISH thus provides a reliable means for prenatal detection of balanced or unbalanced chromosome 12 rearrangements in this family. This approach is possible for identification of similar chromosome rearrangements provided that probes for the putatively involved chromosome region are available.

Molecular cloning and sequencing of the murine alpha-2-macroglobulin receptor cDNA.

We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.

Assignment of the gene coding for the alpha 2-macroglobulin receptor to mouse chromosome 15 and to human chromosome 12q13-q14 by isotopic and nonisotopic in situ hybridization.

The assignment of the gene encoding the alpha 2-macroglobulin receptor (A2MR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-D1. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome 12q.