Frank H. Ruddle

The Human Transferrin Receptor Gene: Genomic Organization, and the Complete Primary Structure of the Receptor Deduced from a cDNA Sequence

Heteroduplex analysis shows that the transferrin receptor gene contains at least 19 distinct coding sequences distributed over 31 kb of genomic DNA. The nucleotide sequence of these coding regions has been determined from a cDNA clone. The sequence contains a single complete open reading frame of 2280 bases which specifies a 760 residue polypeptide with a molecular weight of 85K daltons. The deduced amino acid sequence of the receptor shows that it does not contain an N-terminal hydrophobic signal peptide. We have found a single region of sufficient length and hydrophobicity to span the membrane, located 61 amino acids from the N-terminus. This leads to the prediction that the receptor is oriented in the membrane with a cytoplasmic N-terminus and an extracellular C-terminus. The receptor has no significant homology with transferrin, or with any receptor for which a sequence is available.

A review of enzyme polymorphism, linkage and electrophoretic conditions for mouse and somatic cell hybrids in starch gels.

The selective loss of human chromosomes from both mouse + human and Chinese hamster x human somatic cell hybrids makes these two systems useful for studying problems in gene regulation and gene mapping. The loss of a particular human chromosome may be correlated with the loss of a human enzyme from the hybrid as demonstrated by gel electrophoresis permitting the mapping of the corresponding gene. The use of human translocation cell lines as one of the parental lines in the hybrids makes it possible to map genes to specific sections of the human chromosomes as Ricciuti and Ruddle (34) have recently done for phosphoglycerate kinase (PGK), hypoxanthine guanine phosphoribosyl transferase (HGPRT), and glucose 6-phosphate dehydrogenase (G6PD). We review here the polymorphism and electrophoretic phenotypes and staining conditions for 30 enzymes which may be examined in somatic cell hybrids of’ mouse, human or Chinese hamster cell lines. In most instances the genes for these enzymes have been mapped.

Molecular cloning and chromosome mapping of a mouse DNA sequence homologous to homeotic genes of drosophila

Some of the homeotic genes of Drosophila, involved in the control of segmental development, form a diverged multigene family. A conserved DNA sequence common to these genes has been used to isolate a clone (Mo-10) from the mouse genome which contains a sequence coding for a protein domain that is homologous to the domain conserved in the Drosophila homeotic genes. By structural analogy, this sequence may be involved in the control of metameric pattern formation in the mouse. Mo-10 has been mapped to the proximal portion of mouse chromosome 6, and its position in relationship to genes known to influence mouse morphogenesis is discussed.

Genetic control of two electrophoretic variants of glucosephosphate isomerase in the mouse (Mus musculus)

The autosomal variation and the genetic control of GPI has been determined by a comparison of electrophoretic patterns of F1 and backcross progeny of three inbred strains of mice. The locus controlling the production of GPI in the mouse has been designated Gpi-1. Two alleles at this locus have been described and designated Gpi-1a and Gpi-1b, which represent, respectively, the slow and fast electrophoretic forms. Twenty-seven inbred strains of mice have been classified for these two alleles. The absence of close linkage of Gpi-1 to seven other genetic loci has been determined. It has been demonstrated that the polymorphism of Gpi-1 is widely distributed in feral mice. GPI was expressed in vitro and in four types of malignant tumors.

Localization of the human α-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

Abstract We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.

Chromosomal abnormalities in the human population: estimation of rates based on New Haven newborn study.

The incidence of gross chromosomal abnormality was measured in a large (4500), relatively unbiased sample of New Haven infants born during 1 year. The frequency of infants with abnormal chromosomal constitutions was 0.5 percent. For mothers over age 34, 1.5 percent of newborns were chromosomally abnormal. Only one in four of these infants could have been detected by phenotypic criteria alone. Methods are discussed whereby this fraction of the newborn population might be detected and possibly reduced.

Expression of liver phenotypes in cultured mouse hepatoma cells: Synthesis and secretion of serum albumin

Abstract A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones. The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.

Hox cluster duplications and the opportunity for evolutionary novelties

Hox genes play a key role in animal body plan development. These genes tend to occur in tightly linked clusters in the genome. Vertebrates and invertebrates differ in their Hox cluster number, with vertebrates having multiple clusters and invertebrates usually having only one. Recent evidence shows that vertebrate Hox clusters are structurally more constrained than invertebrate Hox clusters; they exclude transposable elements, do not undergo tandem duplications, and conserve their intergenic distances and gene order. These constraints are only relaxed after a cluster duplication. In contrast, invertebrate Hox clusters are structurally more plastic; tandem duplications are common, the linkage of Hox genes can change quickly, or they can lose their structural integrity completely. We propose that the constraints on vertebrate Hox cluster structure lead to an association between the retention of duplicated Hox clusters and adaptive radiations. After a duplication the constraints on Hox cluster structure are temporarily lifted, which opens a window of evolvability for the Hox clusters. If this window of evolvability coincides with an adaptive radiation, chances are that a modified Hox cluster becomes recruited in an evolutionary novelty and then both copies of duplicated Hox clusters are retained.

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse.

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.

Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes

Constitutive heterochromatin of chromosomes can be visualized utilizing a new differential staining technique which was originally developed by Gall and Pardue (1971). The method facilitates the more certain identification of specific chromosomes within and between cell populations of different origins. Marker chromosomes can be identified in established cell lines over many months of serial passage. Chromosomes of similar morphology within karyotypes of man and mouse can be distinguished in a number of instances. For example, the Y chromosomes of both mouse and man can now be easily detected. The hetero-chromatic staining method also permits discrimination between mouse and human chromosomes in somatic cell hybrids, thus facilitating the assignment of gene markers to chromosomes in somatic cell genetics systems. Instances of translocation of centric heterochromatin to other parts of chromosomes in established tissue culture cell lines are described. An instance of the inheritance of a polymorphic variation in autosomal heterochromatin in man is reported. It is postulated that polymorphisms in the centric heterochromatin may account largely for small heritable chromosome length variations previously described in human populations and termed minor chromosome variants.