Biomedical subjects

G Amore

Publications and source records attributed to G Amore.

14 recordsLinked to original sources

PubMed2000

Recovery of developmentally defined gene sets from high-density cDNA macroarrays.

New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as approximately 300 sea urchin embryos, or 2 x 10(5) cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.

Animals↗

PubMed1998

Effect of the IMPase inhibitor L690,330 on sea urchin development.

A variety of concentrations of the IMPase inhibitor L690,330 were added to sea urchin embryos. Immediate arrest of development was obtained for concentrations from 7.5 mm on. Concentrations lower than 3.5 mm permitted gastrulation but inhibited skeletogenesis and disturbed elongation along the animal-vegetal axis. The latter results are similar to those obtained by counteracting lithium effect with myoinositol, which are suggested to be due to partial relief of IMPase inhibition.

5'-Nucleotidase↗

PubMed1997

Identification and characterization of a constitutive HSP75 in sea urchin embryos.

An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchin P. lividus. This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasms but not in the nuclei. In the embryos a higher hsp75 concentration was found in the portion facing the lumen of the cells which invaginate at gastrulation.

Amino Acid Sequence↗

PubMed1995

Activation by heat shock of hsp70 gene transcription in sea urchin embryos.

We had previously shown that P. lividus embryos subjected to heat shock are unable to synthesize proteins of the hsp70 family at a detectable level before the hatching blastula stage. We show here that this is not due to the inability to synthesize the hsp70 mRNAs, which are detectable following heat shock also in early stages, although in much lower amounts per embryo than in later stages. These mRNAs are also translated, as judged by the facts that they are present in the polysomal pellet, and that they are translatable in a cell free system. As to the question of the amount of hsp70 RNAs per nucleus, we conclude that this is also higher in later than in earlier stages. The presence of hsp70 mRNAs is already detectable after heating at 4 centigrades above 20 and their amount increases with the increase of temperature in the range between 24 degrees C and 28 degrees C.

Animals↗

PubMed1970