Molecular phylogeny in 3-D.

Molecular phylogenetic trees are constructed in three dimensions relative to the distribution of MW and pl classes and immunocrossreactivity against polyclonal antibodies to lens crystallins, as well as multiple sequence alignment between amino acid sequences, coding nucleotide sequences and the gene nucleotide sequences for beta-globin. Euclidian distances are estimated to position species in x, y, z space by multidimensional scaling and merged with bootstrap-tested branching pattern of Fitch & Margoliash plots to obtain 3-D phylogenetic tree. Compared to single attributes, phylogenetic trees based on multiple parameters allow significant repositioning of rodents, chiroptera and primates.

Expression of nerve growth factor during the development of nervous system in early chick embryo.

In the chick gastrula, nerve growth factor (NGF) is localized to the endoblast mesoblast presumptive head ectoderm but not in the presumptive neuroectoblast. During early morphogenesis the dorsal body ectoderm presumptive neural crest cells exhibit strong NGF positive cell surface reaction. NGF appears to be a marker of cells participating in morphogenetic movements but not early neural differentiation. NGF is localized where neural folds fuse and cells die allowing detachment of the neural tube from head ectoderm as well as in dead cells in the neurocoele. NGF reactivity in cells lining the evaginated extremities of the optic vesicle the floor of the neural tube the splanchnopleure heart primordia the inner outer surfaces of somites is suggestive of the role of NGF in primitive organ shaping.

The neural inductive response of competent chick ectoblast decreases away from the host axis and correlates with an increased proliferative activity.

We have assessed the quality and quantity of the neural inductive response of the chick gastrula ectoblast located at increasing distancefrom the host axis. In a stage 4 gastrula, entire ectoblast exhibits neural competence. The quality of induced neural tissue shifts from deuterencephalic type in the area pellucida to archencephalic type in the area opaca and primitive medullary or palisade type atthe margin of overgrowth with a concomitant reduction in the number of induced neural cells. In contrast, the mitotic and 3H-TdR labelling frequencies in the competent ectoblast increase with increasing distance from the host axis and in a proportion inverse to the amount of induced neural tissue. It is suggested that the strong neural inductive response is correlated with low proliferative activity, or longer cell cycle time, of the competent ectoblast.

The neural inductive signal is transferred to ectoblast in 1-2 h but a continued contact with mesoblast for 2-3 h is essential for neuralization in the chick area pellucida.

In the area pellucida of the chick gastrula, the Hensen's node (HN) graft must contact the competent ectoblast for at least 4 h to promote neural induction. When we removed the grafted HN after 1 to 3 h and replaced it by a non-inducing post nodal (PN) fragment, a 1-2 h contact with HN was found to be sufficient to promote neural induction. When HN graft was removed after 3 or 4 h and replaced by PN, the neural inductive response was substantially improved towards formation of archencephalic structures. Thus, our results indicate that neural induction takes place in two steps. In the first step, a contact with HN for 1-2 h is sufficient to transferthe inductive signal which is stabilized through a second step involving continued cell-cell contact with even non-inducing PN mesoblast.

Genetic evidence for posterior specification by convergent extension in the Xenopus embryo.

Genetic studies substantiate that mesodermal convergent extension expressed behind the anteroposterior borderline, in the form of a gradient with the posterior apex after gastrulation, regulates morphogenesis of the posterior zone at the dorsal and dorso-lateral levels which is in full agreement with the model of dorsalization-caudalization. In contrast, how anteroposterior specification of mesodermal tissues occurs at the ventral and latero-ventral levels is not yet understood.

Lens protein phylogeny: immunocrossreactivity of vertebrate lens antigens to anti-shark crystallin antibody.

Antisera prepared against total water-soluble lens proteins of the shark, Scoliodon sorrakowah were reacted with homologous antigen and analysed reaction products by immunoelectrophoresis (IE) and two dimensional crossed antigen-antibody electrophoresis (2D-CE). On IE, shark antigens formed 5 precipitin lines including 1 alpha, 3 beta and 1 gamma crystallins and on 2D-CE 3 alpha, 6 beta and 6 gamma peaks accounting for 8%, 27% and 65% antigen in the respective group were obtained from the total crystallins. Using anti-shark antisera, the immunocrossreactivity of lens proteins from 6 Chondropterygii, 23 teleosts and 16 higher vertebrates was examined by IE. It is found that beta crystallins are the most conserved and crossreact with all vertebrate classes, whereas gamma crystallin crossreactivity is specific to the class Pisces and alpha crystallins are least conserved and their crossreactivity is confined to subclass Chondropterygii. Based on IE patterns, a phylogenetic tree is constructed which demonstrates the intrafamily closeness except in case of adaptive radiation.

Inhibition of cleavage by restriction endonucleases due to modifications induced in SV40 DNA by methyl methanesulfonate.

Methyl methanesulfonate (MMS), a direct mutagen, methylates DNA bases and causes distortions in DNA structure. Supercoiled SV40 DNA was treated in vitro with varying concentrations of MMS from 0.001 mM to 10 mM MMS either for 30 min or 3 h and analysed by electrophoresis in 1% neutral and alkaline agarose gels. The electrophoretic mobility (EPM) of native DNA did not change after treatment with the mutagen, while alkaline gels revealed low MW DNA fragments due to single strand breaks at alkali-sensitive sites generated by the action of MMS. By two-dimensional electrophoresis, we find that all three native DNA forms contain alkali-sensitive sites after treatment with MMS. To examine the effect of base modification by MMS on DNA-protein interactions, we have used as probes, restriction endonucleases. These cleave DNA in a sequence-specific manner, and their activity is dependent upon the methylation status of the substrate DNA. We find that cleavage by these restriction endonucleases is inhibited due to methylation by MMS.

Fast inducible repair of microinjected UV-irradiated SV40 DNA in monkey kidney cells.

In monkey kidney cells (TC-7), microinjected with UV-irradiated (103-362 J/m2) SV40 DNA, the expression of viral antigens decreases in a UV-dose-dependent manner and the viral genes are not repaired constitutively. When the viral DNA is microinjected 4 h after UV-irradiation (40 J/m2) of host cells, the expression of viral antigens is restored in all cells. The time course of restoration of viral gene expression function shows that in UV-irradiated cells the repair is induced rapidly and fully within 2 h and the induced state is maintained for 24 h. Intact viral DNA molecules, microinjected during the period of induction of cellular UV repair, are expressed less efficiently than UV-irradiated viral genomes.

Caudalization by retinoic acid is correlated with inhibition of cell population growth and expansion of chick blastoderms cultured in vitro.

Full primitive streak stage chick embryos, cultured in vitro for 20 h in the presence of 10(-9) to 10(-7) moles of retinoic acid (retinol, all-trans), exhibit increasing extent of malformations. RA causes caudalization, suppression of telencephalon, formation of open and enlarged neural tube in the regions of midbrain, hindbrain and spinal cord, failure of fusion of heart tubes, and gives rise to winged or diffused, and even supernumerary somites. Extreme abnormalities include failure to form the head fold and foregut. Abnormal embryos were graded according to Rao and Chauhan (Teratology 4: 191-198, 1971), and we find that the larger the severity of malformation, the smaller the size of total cell population and blastoderm area. Concomitant to caudalization, retinoic acid suppresses the cell population growth.

Multiple neural inductions in area opaca by grafts of the Hensen's node do not retard host chick embryo development.

Four hundred and forty four full primitive streak stage chick embryos were cultured in vitro and 261 were transplanted with 1, 3 or 5 Hensen's nodes in the area opaca. Irrespective of the number of grafts, neural induction was observed in 90% cases. The development of control and grafted embryos and the size of blastoderm area were monitored at the time of grafting and after 20 hr. We find that the induced neural tissue and differentiated tissue of graft-origin neither fuse with the host embryonic axis, nor retard its development.

Regulation of Xenopus c-myc promoter activity in oocytes and embryos.

We have studied the regulation of transcription of the Xenopus c-myc I gene in oocytes and embryos. Various 5' and internal deletions of a 1310-bp-long c-myc I promoter fragment have been ligated upstream of the chloramphenicol acetyl transferase (CAT) reporter gene and microinjected into oocytes and fertilized eggs. Activity was determined by CAT assay and primer extension. The c-myc promoter drives transcription very efficiently, and a truncated promoter -158/+46 essentially retains full activity. This region contains an overlapping E2F/SP1 site and two tandem Sp1 sites homologous to those found in the c-myc gene of mouse. Internal deletions show that both elements are equally active in oocytes in driving the expression of CAT. A germinal vesicle extract contains a DNA-binding activity specific for an Sp1 consensus sequence but not the E2F site. The data suggest that the high transcription level of the endogenous c-myc gene in Xenopus oocytes is mediated by Sp1 or a related transcription factor. In embryos a different mechanism emerges and the functional role of the Sp1 binding sites appears to be less important.

Physicochemical characterization and phylogenetic comparison of fish lens proteins.

The composition of soluble eye lens proteins from four chondropterygiian and fourteen teleostean fishes were analyzed for heterogeneity in MW and pI. Lens proteins from all the fish species studies are distributed in the pI range 4.3-9.0 with polypeptides in the range 17,500-31,000 Da. Phylogenetic trees are constructed based on the observations.

Rat liver chromatin digested in situ with endogeneous Mg(2+)-dependent deoxyribonuclease exhibits two distinct DNA repeats.

Incubation of rat liver nuclei in the presence of 1.0-5.0 mM Mg2+ at 37 degrees C releases oligonucleosomes containing at least two distinct chromatin-DNA repeat elements. The 'short' repeat is derived from the dimer to pentamer series, while the 'long' repeat is found in the monomer and hexamer to decanucleosomes. Both repeat lengths decrease during enzymatic hydrolysis but in 5.0 mM Mg2+, which is optimal concentration, the 'long' repeat is degraded faster.

Isonicotinic acid hydrazide inhibits cell population growth during teratogenesis of chick embryo.

In chick embryos treated with a 4 hr pulse of 7.2 X 10(-5) M isonicotinic acid hydrazide (INH) the cell population growth is inhibited with an increased population doubling time. Teratogenised blastoderm cells complete their ongoing cell cycle and arrest in G1 phase. A chase with an equimolar concentration of pyridoxal-5-phosphate restores the growth rate after a lag of 4 hr equivalent to the duration of treatment with INH. Presumptive mesoblast cells invaginated through the primitive streak and neuroectoblast cells induced prior to the application of INH differentiate, while the teratogen inhibits morphogenesis and organization of organ primordia.

Cell population growth in chick blastoderms cultured in vitro.

Primitive streak stage chick blastoderms were cultured in vitro up to 30 hr by New's technique. Chick blastoderms reaching stages 4 to 12 in vitro cultures and in ovo were harvested and homogenized to release cell nuclei. Fluorescent ethidium bromide-stained nuclei in homogenates were counted in Neubauer's chamber and the size of total blastoderm cell population was determined. Linear regression analysis revealed that both in ovo and in vitro chick blastoderm cell population grows in a biphasic manner with comparable cell population doubling times and the morphogenesis is not affected in vitro during the culture period.

Nuclear endogenous Ca2+-dependent endodeoxyribonuclease in differentiating chick embryonic lens fibers.

During terminal differentiation of lens epithelial cells into fiber cells, nuclei become pycnotic and DNA degradation occurs. We investigated the putative role in this process of an endogenous DNAase. After incubation of isolated nuclei of both cell types at 37 degrees C, DNAase activity was revealed by DNA size analysis on 0.3-1% neutral and alkaline agarose, one- and two-dimensional gels. This DNAase activity is more prominent in lens fiber nuclei than in epithelial nuclei at all the embryonic stages probably because of a preexisting higher concentration of divalent cations in the former. This activity is calcium or magnesium dependent in both types of nuclei.

DNA repeat size in chick embryonic lens epithelium, lens fiber, brain and liver cell nuclei.

The DNA repeat size is determined by micrococcal nuclease digestion kinetics and subsequent electrophoresis of the products among various chick embryonic tissues. The repeat size is found to be not significantly different from 193 to 197 bp, for brain and liver at 11 days and for lens epithelium and fiber at different embryonic stages. However, the pattern of micrococcal digestion seems to reveal an overall chromatin modification as a function of development in the lens fibers.

Immunochemical characterization and quantitative distribution of crystallins in the epithelium and differentiating fibre cell populations of chick embryonic lens.

Lenses from 19-day chick embryos are fractionated by a double punch method to obtain the epithelium-annular pad complex (EP), outer fibres (OF), middle fibres (MF) and central fibres (CF). Water-soluble crystallins are characterized by SDS PAGE, isoelectric focusing (IEF) and two-dimensional IEF-SDS PAGE. Crystallins are also characterized by immunoelectrophoresis (IE), rocket IE, IEF-immunoblotting, and quantified by two-dimensional antigen-antibody crossed electrophoresis using antibodies to total 19-day embryonic as well as adult crystallins. In the adult lens, alpha-, beta- and delta-crystallins are 19%, 67% and 14%, respectively, while these are present at concentrations of 9%, 27% and 64%, respectively, in 19-day embryonic lens. In absolute amounts, delta-crystallin increases only by 1.23-fold between 19-day embryonic age and 6 months post-hatching, while total lens protein increases 12.5-fold. The predominance of delta-crystallin in central fibres, located along the optical axis, suggests that this protein is of embryonic origin. delta-Crystallin from fibres is electrofocused as 12 distinct molecular classes (pI 5.2-5.42) which react against anti-delta-crystallin on an immunoblot. Of these, the three most anodal species are not detected in EP. Fibres contain 50 000, 48 000 and 45 000 dalton delta-crystallin subunits while only 50 000 and 48 000 dalton subunits are present in EP.