Alan Y. Sakaguchi

Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes

The human R-ras gene was isolated by low-stringency hybridization with a v-H-ras probe. The predicted 218 amino acid R-ras protein has an amino-terminal extension of 26 residues compared with H-ras p21, and shows 55% amino acid identity; conserved domains include the p21 GTP-binding site and the carboxy-terminal membrane localization sequence. R-ras has at least six exons, with the position of the first intron conserved relative to the Drosophila ras64B and Dictyostelium ras genes; there is no similarity in the exon-intron structure of the R-ras gene and of the mammalian H-, K-, and N-ras proto-oncogenes. Cloned mouse R-ras cDNAs exhibit 88% nucleotide and 94.5% predicted amino acid identity to human R-ras. Human R-ras was localized to chromosome 19, a site different from ras p21 genes. Mouse R-ras is syntenic with c-H-ras on chromosome 7.

Mapping the human genome, cloned genes, DNA polymorphisms, and inherited disease.

It is estimated that the human haploid genome is composed of 3 × 109 nucleotides and that only 10% of it consists of coding and regulatory sequences.14 If a gene is approximately 104 nucleotides in length, which includes the coding region and the intervening and flanking sequences, this estimate would predict that there are about 3–10 × 104 human genes coding for different protein products. Since gene clustering in humans has become evident (for example, the hemoglobin, immunoglobin, and HLA clusters), these estimated gene products may be grouped in from 3000 to 15,000 clusters.15 Further, based upon genetic and molecular studies of microorganisms, Drosophila, and the mouse, there are perhaps 5 × 104 structural genes estimated in humans,14,106,183 which is in agreement with the number of estimated protein products in the human genome. Mapping the human genome involves partitioning the total number of genes into individual maps representing the 24 different human nuclear chromosomes and linearly ordering them on each chromosome. A similar exercise has mapped the 37 genes encoded in the DNA of the mitochondrial genome.2

Human ferritin H and L sequences lie on ten different chromosomes

SummaryIn humans, the H (heavy) and L (light) chains of the iron-storage protein ferritin, are derived from multigene families. We have examined the chromosomal distribution of these H and L sequences by Southern analysis of hybrid cell DNA and by chrosomal in situ hybridization. Our results show that human ferritin H genes and related sequences are found on at least seven different chromosomes while L genes and related sequences are on at least three different chromosomes. Further, we have mapped the chromosomal location of expressed genes for human H and L ferritin chains and have found an H sequence which may be a useful marker for idiopathic hemochromatosis.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Human apolipoprotein A-I and C-III genes reside in the p11 → q13 region of chromosome 11

Apolipoprotein (apo) A-I is a major protein of high density lipoproteins (HDL). The gene for apoA-I has been localized to the p11 leads to q13 region of chromosome 11 by filter hybridization analysis of mouse-human hybrid cell cDNAs containing chromosome 11 translocations utilizing a cloned human apoA-I cDNA probe. The known linkage of apoA-I and apoC-III also permitted the simultaneous assignment of the apoC-III gene to the same region on chromosome 11. Comparison with previously established gene linkages on the mouse and human genome suggests that apoA-I + apoC-III may be linked to the esterase A4 and uroporphyrinogen synthase genes which are present on the long arm of human chromosome 11. The localization of the apoA-I + apoC-III genes in the p11----q13 region of chromosome 11 represents a definitive chromosomal assignment of a human apolipoprotein gene, and will now enable more detailed analysis of the geneomic organization and linkages of the apolipoprotein genes.

The DNF15S2 locus at 3p21 is transcribed in normal lung and small cell lung cancer.

Small cell lung cancer (SCLC) has been associated with a deletion of the short arm of chromosome 3. One SCLC cell line, H748, has an interstitial deletion of chromosome 3p and shows allele loss for the DNF15S2 locus detected by the probe lambda H3. Conservation of DNF15S2 sequences in mouse indicated that this human genomic fragment may contain coding sequences. Screening of a normal lung cDNA library with chromosome 3-specific fragments of the lambda H3 probe resulted in the isolation of 18 positive clones. The cDNA clones detect an additional DNA polymorphism that is in linkage disequilibrium with the HindIII polymorphism of the DNF15S2 locus. Sequence analysis indicated that the DNF15S2 locus could potentially code for a previously unreported protein of 67 kDa which has 26 cysteine residues. DNF15S2 is part of the coding region of a 3.3-kb mRNA expressed in lung. Northern analysis indicated that this mRNA was not detectable in one of five SCLC lines. This SCLC line, H128, also lacks the enzyme aminoacylase 1.

Regional localization of two human cellular Kirsten ras genes on chromosomes 6 and 12

Human cellular Kirsten ras1 and ras2 genes were localized to chromosomes 6p23 ----q12 and 12p12 .05----pter, respectively, using human-rodent cell hybrids. Thus, the short arms of human chromosomes 11 (encoding lactate dehydrogenase-A and the proto-oncogene c-Ha- ras1 ) and 12 (encoding lactate dehydrogenase B and c-Ki- ras2 ) share at least two pairs of genes that probably evolved from common ancestral genes.

The human apolipoprotein A-II gene is located on chromosome 1

Apolipoprotein (apo) A-II is a major constituent of high density lipoproteins (HDL). The gene for apoA-II has been localized to the p21----qter region of chromosome 1 in man by Southern blot hybridization analysis of DNA from human-mouse cell hybrids using a cloned human apoA-II cDNA probe. The regional assignment was established using two hybrids carrying a reciprocal translocation involving chromosomes 1 and 2. Comparison with previously established gene loci on chromosomes 1 suggests that apoA-II may reside in a conserved linkage group with renin and peptidase C. On the other hand, apoA-II is not linked to the apoA-I gene, which has been localized previously to chromosome 11.

Mapping and conservation of the group-specific component gene in mouse

The group-specific component (GC), also known as the vitamin D-binding protein, transports vitamin D and its metabolites in plasma to target tissues throughout the body. The GC gene shares an evolutionary origin with genes encoding albumin (ALB) and alpha-fetoprotein (AFP). All three genes are descendants of an evolutionary ancestor that arose from an intragenic triplication. As a result, each gene is composed of three homologous domains. The study described here characterizes and compares mouse GC to the corresponding nucleotide and amino acid sequences of GC from human and rat. The deduced amino acid sequence of mouse GC was 78% identical to human and 91% identical to rat GC. The results suggest that, unlike the corresponding sequences in the ALB and AFP genes, chromosomal sequences encoding the first domain and the leader sequence of the GC gene have specifically been conserved throughout vertebrate evolution. Protection of domain I during evolution may correlate with an important functional aspect of its sequence. The mouse GC gene was mapped to chromosome 5, where the ALB and AFP genes are also located, demonstrating conservation of the three genes in vertebrate species.

Chromosome assignment of mouse insulin, colony stimulating factor 1, and low-density lipoprotein receptors

Receptors for insulin, low-density lipoprotein, and colony stimulating factor 1 are associated with diabetes, atherosclerosis, and cancer in man. Complementary DNA clones for Insr, Ldlr, and Csfmr were used to chromosomally assign the three genes in mouse. In contrast to their close linkage on the short arm of human Chromosome 19, Insr and Ldlr are asyntenic, residing on mouse Chromosomes 8 and 9, respectively. The genes for CSF1R, CSF1, CSF2, IL-3, and IL-5 form a cluster on the long arm of human Chromosome 5. In mouse, Csfm, Csfgm, and IL-3 are syntenic on Chromosome 11. The Csfmr gene was assigned to mouse Chromosome 18 and is thus unlinked to other members of this gene cluster. These gene assignments provide additional topographical information on conservation of linkage groups in man and mouse and provide a genetic framework for evaluating the possible roles for the three receptor genes in genetic diseases in mouse.