Carl Hilliker

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.

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.

Assignment of the gene coding for the α2-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.

Human and rat chromosomal localization of two genes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by analysis of somatic cell hybrids and in situ hybridization

Two genes encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were localized in human and rat chromosomes. PFKFB1 (previously PFRX), which encodes the liver and muscle isozymes, was assigned to Xq22-q31 in the rat and to Xq27-q28 in the human by in situ hybridization using probes generated by the polymerase chain reaction. PFKFB2, which encodes the heart isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was assigned to chromosome 13 in the rat and to chromosome 1 in the human by hybridization of DNA from somatic cell hybrids. By in situ hybridization, this gene was localized to the regions 13q24-25 in the rat and 1q31 in the human.

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 Hodgkins 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.

Localization of the gene encoding the α2 subunit of the human VLA-2 receptor to chromosome 5q23-31

The α2 subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the α2 subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-α2 antibody 12F1. Intracellular α2 antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic α2 sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published α2 cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying aCD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a3H-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization ofCD49B on chromosome 5 at q23-31.

MOLECULAR CYTOGENETIC ANALYSIS OF A FAMILIAL PERICENTRIC-INVERSION OF CHROMOSOME-12.

Speleman F, Van Roy N, De Vos E, Hilliker C, Suijkerbuijk RFS, Leroy JG. Molecular cytogenetic analysis of a familial pericentric inversion of chromosome 12

Cloning, characterization, and chromosomal localization to 4p16 of the human gene (LRPAP1) coding for the α2-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.

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.

Characterization of the Genes Coding for the Murinoglobulins and Expression in Vivo

Proteinase inhibitors of the a-macroglobulin (aM) family all have the typical feature of being activated by proteolysis in the bait region and by reaction at the cysteinyl glutamyl internal thiol ester site. Most are tetramers, but dimeric and monomeric forms have been identified. Monomeric or-macroglobulins have been described in the plasma of rodents and, more recently, also in amphibians, reptiles, and birds. In mouse plasma, two different types of a M have been described: the tetrameric aZM (MAM) and the monomeric murinoglobulins (MUG). Three different MUG cDNA clones were isolated and sequenced, of which two contained a full-length open reading frame, whereas the third was aberrant. This contrasts with the fact that, in mouse plasma, only one protein was isolated and N-terminally sequenced, corresponding to the most abundant cDNA clones obtained, designated MUGl. We have now characterized the family of MUG further at the genomic level, concentrating on the bait region, which is both structurally and functionally the most specific region. Lambda clones representing genes coding for four different murinoglobulins were isolated. In each gene, the bait region was encoded by two exons (numbered 17 and 18 in analogy to the structure of the MAM gene determined by us), with a sequence that unequivocally established the identity of three of the four genes as coding for the three MUG cDNA types cloned previously. Sequencing of the exon-intron boundaries, intron sizing by PCR, and restriction analysis resulted in physical maps of the region encompassing exons 15 to 25 in each MUG gene. Southern blotting of the DNA of five different mouse strains and one intercross confinned this and suggested even the presence of a fifth murinoglobulin gene. The exon and intron sequences obtained have shown that the actual bait region sequences are much more divergent than the exons coding for protein domains flanking the bait. Even the intron sequences adjacent to the bait exons are more conserved than the bait sequences themselves. This strongly suggests positive Darwinian selection as the mechanism for divergence of the MUG bait region, generating genes coding for proteinase inhibitors with potentially novel inhibitory properties. The expression of MAM and MUG was examined during adolescence and during pregnancy by Northern blotting of liver mRNA and by immunoelectrophoresis of mouse plasma. The level of circulating MAM observed at birth was maintained into