Makoto Okazaki

Recombinant Human Growth/Differentiation Factor 5 Stimulates Mesenchyme Aggregation and Chondrogenesis Responsible for the Skeletal Development of Limbs

We have expressed and biologically characterized recombinant human growth/differentiation factor 5 (huGDF5). This protein is composed of a mature homodimer consisting of 15 kD subunits. Using recombinant expressed protein, we have demonstrated that huGDF5 in vitro stimulated mesenchyme aggregation and chondrogenesis in rat limb bud cells. In vivo, partially purified huGDF5 induced cartilage and bone formation in muscular tissues of rodents. However, in contrast to the effects of other BMPs, as for example BMP-2, the osteoblastic MC3T3-E1 cells did not respond to huGDF5 as measured by alkaline phosphatase activity. These results suggest that the action of GDF5 may be relatively specific for chondrogenesis during the entire process of the endochondral bone formation. GDF5 may control the morphogenesis of cartilaginous tissue, including joints, in the skeletal development of limbs.

Loss of alleles at loci on chromosome 13 in human primary gastric cancers.

Mitotic events leading to the loss of the normal allele corresponding to a mutated gene are important for tumorigenesis in rare heritable tumors such as retinoblastoma and Wilms tumor. As reported for both colorectal and breast cancers, some common tumors seem to develop because of the same mitotic events. We examined constitutional and tumor genotypes defined by polymorphic DNA clones in 36 patients with gastric cancer. In 14 cases, constitutional heterozygosity at loci on chromosome 13 had been lost. Loss of alleles was also detected at a locus on chromosome 18 in two cases and at a locus on chromosome 17 in one case. The frequent loss of alleles at loci on chromosome 13 (41%) suggests that elimination of genes on this chromosome may be of importance in the tumorigenesis of human primary gastric cancers.

The NH2-terminal region of the active domain of sonic hedgehog is necessary for its signal transduction

The NH2‐terminal domain of sonic hedgehog (residue 25–198) was expressed in both yeast and animal cells. The yeast‐derived NH2‐terminal domain of sonic hedgehog was less active by far than the animal cell‐derived counterpart. The yeast‐derived NH2‐terminal domain of sonic hedgehog lacked 10 amino acids from the NH2‐terminus. This cleavage of the yeast‐derived NH2‐terminal domain of sonic hedgehog might due to Kex 2. In contrast, a mutant yeast‐derived NH2‐terminal domain of sonic hedgehog (Lys‐33 to Thr) retained its NH2‐terminus and its activity was comparable to that of the animal cell‐derived NH2‐terminal domain of sonic hedgehog. The NH2‐terminal deleted NH2‐terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell‐derived NH2‐terminal domain of sonic hedgehog in a dose‐dependent manner. These data suggest that the NH2‐terminal deleted NH2‐terminal domain of sonic hedgehog retains a receptor‐binding ability and that the NH2‐terminal peptide of the NH2‐terminal domain of sonic hedgehog is necessary for its signal transduction.

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

SummaryThe gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.

The zygosity determination of Japanese twins using a minisatellite core probe

SummaryHypervariable ‘minisatellite’ regions which are dispersed in the human genome show restriction fragment length polymorphisms (RFLPs) due to allelic differences in the number of tandem repeats containing the core sequence. Southern blot hybridization using minisatellite core probes produces various band patterns, which are completely individual-specific and of which few fragments are shared between two randomly selected individuals. If the band patterns are identical between twins, they must be monozygotic. We report here the use of minisatellite core probe for zygosity determination in five Japanese twin pairs and in a set of triplets.

Assignment of a polymorphic locus of OS-4(D18S5) DNA segment to human chromosome region 18q21.3→qter

SummaryA polymorphic human DNA fragment, OS-4, isolated from pBR322 human genomic library was mapped to chromosome 18 using a human-mouse somatic cell hybrid panel. More precise assignment of this locus to 18q21.3→qter was made by hybridization to DNA from six cell lines containing different structural abnormalities of chromosome 18.In addition to TaqI polymorphism, it was proved that OS-4 could detect polymorphism in PstI-digested human DNA. This probe, designated as D18S5, would be a useful marker for gene mapping as well as linkage analysis of genetic diseases.

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Preclinical detection of men 2A gene carrier using linked DNA markers

SummaryWe have performed preclinical risk estimation of multiple endocrine neoplasia type 2A (MEN 2A) by using the polymorphic DNA markers tightly linked to the MEN2A locus. The gene for MEN 2A has been assigned to the pericentromeric region of chromosome 10 by linkage analysis. The preclinical detection of gene carriers in MEN 2A families using tightly linked DNA markers is useful for surgical treatment at an early stage. The DNA markers, RBP3 (retinol-binding protein 3, interstitial) and FNRB (fibronectin receptor, beta polypeptide), are both tightly linked to the MEN2A locus, and are localized to opposite sides of the MEN2A locus. We have used RBP3 and FNRB as markers for preclinical diagnosis. Of 18 Japanese families with MEN 2A, 6 families are informative for both loci, and other 10 families are informative for either RBP3 or FNRB. In one informative family, a 20-year-old female is predicted to be the gene carrier (probability; about 99%). She should be carefully followed up till full penetrance age. We conclude that DNA-based prediction of MEN 2A is an effective procedure for clinical use.

Mapping of the genes around MEN2A locus using pulsed-field gel electrophoresis

SummaryThe gene for multiple endocrine neoplasia type 2A (MEN 2A) is closely linked to RBP3 (retinol-binding protein 3, interstitial, probe IRBP.H4) and the DNA marker D10S15 (probe pMCK2), which have been assigned to the proximal long arm of chromosome 10 by linkage analysis both in Caucasian and Japanese populations. We have constructed a rare-cutting restriction map around the RBP3 and D10S15 loci by pulsed-field gel electrophoresis (PFGE). The RBP3 and D10S15 loci appeared to be within a single 160 kb MluI fragment. In 5 patients with MEN 2A, gene rearrangements, such as a gross deletion, were not found in the 880 kb NruI fragment which covered the closest region to the MEN-2A locus from the RBP3 and D10S15 loci.