Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

Cryopreservation of spermatozoa of a transgenic mouse]

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.     

Motor-Substrate Interactions in Mycoplasma Motility Explains Non-Arrhenius Temperature Dependence

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by ∼400 “leg” proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40°C. This corresponds to an Arrhenius factor that decreases from ∼45 k_BT at 10°C to ∼10 k_BT at 40°C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.     

Evolution of gene families and relationship with organismal evolution: rapid divergence of tissue-specific genes in the early evolution of chordates

To determine a possible relationship between organismal and molecular evolution, the divergence patterns of gene families were examined by taking special notice of functional difference, tissue distribution, and intracellular localization of the members. A phylogenetic analysis of 25 different gene families revealed interesting patterns of divergence of these families: Most gene duplications giving rise to different functions antedate the vertebrates-arthropods separation. On the other hand, in a group of members carrying virtually identical function to one another but differing in tissue distribution (tissue- specific isoform), most gene duplications have occurred independently in each of vertebrates and arthropods after the separation of the two animal groups. In family members encoding molecules localizing in cell compartments (compartmentalized isoforms), the gene duplications antedate the animals-fungi separation. In the cases of the Ca2+ pump and rab subfamilies, the compartmentalized isoforms were shown to have diverged during the early evolution of eukaryotes. A phylogenetic analysis of the tissue-specific isoforms from 26 different subfamilies revealed extensive gene duplications and rapid rates of amino acid substitutions in the early evolution of chordates before the separation of fishes and tetrapods. On the contrary, the genetic variations are relatively low in the later period. This pattern of evolution observed at the molecular level is correlated well with that of tissue evolution based on fossil evidence and morphological data, and thus evolution at the two levels may be related.      

Recent gene conversion between genes encoding human red and green visual pigments

Nucleotide sequences of the human X-linked red and green pigment genes were compared, and the number of silent substitutions per site (KSc) between these genes was analysed in comparison with the corresponding values of primate genes. Taking the retarded mutation rate of X-linked genes into consideration (Miyata et al., 1987), the red and green pigment genes were shown to have undergone gene conversion at around the time of separation of African apes and orangutan. Thus the recent gene conversion and retarded mutation rate in these X-linked genes are probably responsible for the strong sequence similarity between these genes, which is likely to facilitate the occurrence of red-green color blindness in the human population. It was also shown that the red pigment gene evolved about five times more rapidly than the green pigment gene since the latest gene conversion.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Cloning and sequence analysis of adrenodoxin reductase cDNA from bovine adrenal cortex

cDNA clones for bovine adrenodoxin reductase were isolated, and the primary structure of the enzyme precursor was deduced from their nucleotide sequences. The precursor consists of 492 amino acids including an extrapeptide of 32 amino acids at the amino terminus. The extrapeptide is hydrophilic [corrected] and rich in arginine. The amino terminal sequence of the precursor is homologous with that of the adrenodoxin precursor. A possible FAD- or NADPH-binding site is present near the amino terminus of the mature enzyme.