Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. I. Isolation and characterization of amphibian tyrosinase mRNA

Rana pipiens tyrosinase mRNA was isolated from Stage 22 (tailfin circulation) embryos by indirect immunoprecipitation of embryonic polysomes using highly specific rabbit anti-tyrosinase and goat-(anti-rabbit) immunoglobulins. Analysis on sucrose gradients indicated that anti-tyrosinase bound specifically to embryonic polysomes of the 300-350 S class coincident with the location of nascent tyrosinase enzyme activity and tyrosinase mRNA. These same anti-tyrosinase-bound polysomes were fully immunoprecipitated by the addition of goat-(anti-rabbit) IgG. Poly(A+) RNA was obtained from phenol-extracted antibody. polysome complexes by sequential passage over oligo(dT)-cellulose. The final purification of tyrosinase mRNA was achieved by preparative sucrose gradient fractionation. Tyrosinase mRNA sedimented as a single 13 S peak in 5-30% sucrose gradients and tracked on sodium dodecyl sulfate-polyacrylamide gels as a single band of 4.5 X 10(5) Da (1275 nucleotides). When assayed in a cell-free translation system, this mRNA directed the synthesis of a single 35,000-Da protein which co-migrated with native tyrosinase on sodium dodecyl sulfate-polyacrylamide gels and which was greater than 98% immunoprecipitable by anti-tyrosinase immunoglobulin. Final purification was 4103-fold over the starting polysomal RNA.     

Preferential expression of Mdm2 oncogene during the development of neural crest and its derivatives in mouse early embryogenesis

The Mdm2 oncoprotein acts as the principal negative regulator of p53 activities and is essential for its control during mouse early development, at least before implantation. We analyzed Mdm2 expression between 7.5 and 9 days post-coitum (dpc) by whole-mount in situ hybridization and report here a novel expression pattern during neural crest development. At 7.5 dpc Mdm2 becomes preferentially expressed at the top of the neural folds. Between 8 and 9 dpc, this preferential expression is also observed in neural crest cells migrating from the closing brain towards craniofacial regions and the first three branchial arches. It persists in the craniofacial mesenchyme and the first branchial arch in 9 dpc embryos. Migrating neural crest cells in the tail region are also preferentially labeled at this stage. At day 9.5 Mdm2 becomes more ubiquitously expressed throughout the embryo as reported before.     

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3'' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.     

Characterization of bone PG II cDNA and its relationship to PG II mRNA from other connective tissues.

Two cDNA clones encoding the small proteoglycan II (PG II) of bone were isolated from a lambda gt11 expression library. These clones expressed recombinant protein which was cross-reactive with polyclonal and monoclonal antisera to PG II molecules from several connective tissues. The longest clone, lambda Pg 20 was studied in detail. The clone was shown to encode PG II by hybrid selected translation and immunoprecipitation. Northern analysis showed two species of the PG II message of approximately 1.4 and 1.8 kb. Substantial amounts of PG II message were found in bone, tendon, articular cartilage, skin, smooth muscle and cornea. Trace amounts of message were also detected in liver and brain. Radiolabeled bovine PG II cDNA hybridized to RNA from several other species including the human, rat and chicken. The level of PG II mRNA in chick embryonic fibroblasts was sensitive to transformation by Rous sarcoma virus.     

Isolation of an abdominal-A gene from the locust Schistocerca gregaria and its expression during early embryogenesis

Using sequence homology to Drosophila homeobox-containing genes, we have cloned a homologue of abdominal-A from the locust Schistocerca gregaria. The Schistocerca clone encodes a stretch of 78 amino acids including the homeodomain and its flanking regions identical to the corresponding region of abdominal-A. We have shown by in situ hybridization that this gene is transcribed and have used an antibody raised against its protein product to examine the expression of abdominal-A during early Schistocerca embryogenesis. Schistocerca is a short germ insect. Although the segmented body plan is very similar to that of Drosophila, the segments are generated sequentially by a process of growth, not simultaneously by subdivision of a syncytial blastoderm. In both organisms, abdominal-A is expressed throughout the abdomen from a sharp anterior boundary located within the first abdominal segment (A1). The initial activation of the genes in the two species differs. Schistocerca initiates expression in a small group of cells in the anterior of A2, shortly after this segment is defined by the appearance of engrailed protein. This contrasts with the appearance of abdominal-A expression in Drosophila, which appears simultaneously throughout the entire abdomen.     

The Mdm2 gene of zebrafish (Danio rerio): preferential expression during development of neural and muscular tissues, and absence of tumor formation after overexpression of its cDNA during early embryogenesis

The Mdm2 protein is most probably the main negative cellular regulator of the p53 tumor-suppressor protein. It was found to be overexpressed in a great number of human tumors and is considered as a potential target for anti-tumor therapies. Mdm2 is an essential gene in mice, yet its role in normal development and tissue differentiation is unknown. In order to study the role of this important protein in an evolutionary perspective, we cloned an Mdm2 cDNA from the fish Danio rerio and analyzed its expression pattern as well as the phenotypic consequences of its overexpression. The main functional domains as well as the interaction between Mdm2 and p53 are conserved in zebrafish. Moreover, we show here that the gene is expressed specifically during early development in neural and muscular tissues. Surprisingly, microinjection of Mdm2 mRNA in two-cell-stage embryos led to inhibition of cellular convergence during gastrulation. The clones derived from Mdm2 microinjected blastomeres were significantly smaller than those derived from control microinjections, and, in contrast to what was observed in Xenopus, did not develop tumors. Our results suggest that Mdm2 expression may be important during the differentiation of neural and muscular tissues of zebrafish. They also point to important differences between phyla in the susceptibility to tumor formation.     

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation.

A cDNA library was constructed in the expression vector lambda gt11, by synthesizing cDNA from size-selected poly(A) RNA from lactating rat mammary gland, using random hexanucleotide primers. Using this library we identified two recombinants which, on addition of a lac z inducer, produced proteins recognized by affinity-purified anti-fatty-acid synthase antibody, and which, therefore, contained fatty acid synthase coding sequences. The inserts were subcloned, were shown to be between 500 and 600 base pairs in size, and to cross-hybridize. The cloned DNA was then used in Northern hybridizations with mRNA isolated at various stages throughout lactation. Two mRNA species were identified of approx. 9.7 and 10.4 kilobases, which increased and decreased in parallel during lactation, reaching a peak at 12-13 days. Both mRNA species disappeared rapidly if the pups were removed prematurely. This study provides evidence that, during hormonal induction in lactation, regulation of the level of fatty acid synthase protein can be accounted for by variation in the level of mRNA.     

The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro.

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.     

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.     

Control of gene expression during induction of cultured peanut cells: mRNA levels,protein synthesis and enzyme activity of stilbene synthase

Stilbene synthase is an inducible enzyme occurring in a small number of plants. The enzyme is amenable to analysis and biochemical studies only after the cells are subjected to induction. Cell suspension cultures of peanut react very selectively if elicited with biotic inducers. Just as intact peanut plants produce stilbene phytoalexins when attacked by fungi so also do sterile cultured cells when treated with sterilized insoluble fungal cell walls. Both systems react by synthesizing stilbene synthase. The time courses of increase in enzyme activity, protein synthesis and mRNA activity were studied, and their relation to other activities of the cells was elaborated. The results show that, after applying the fungal elicitor, the system responds very quickly and selectively: within 2 hours the synthesis rate of stilbene synthase protein is increased more than 30-fold, the increase being detectable 40 min after induction. The first increase in translatable mRNA for stilbene synthase can be seen 20 min after application of the stimulus. Stilbene synthase synthesized in vivo was compared to stilbene synthase prepared by translation in vitro. There was no difference in size, and limited proteolysis did not indicate significant differences in the peptide structure of the primary translation product and the active enzyme.     

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.     

Cis-acting sequences in the 5'-untranslated region of the ribosomal protein A1 mRNA mediate its translational regulation during early embryogenesis of Drosophila.

The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA.