Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer.

A common polymorphism of the early embryonic histone-gene repeat of Strongylocentrotus purpuratus is a 195-bp insertion within the H4-H2B spacer. The sequence, found as an insert in histone-gene repeats of 6 of 22 individuals screened, is also found at approximately 50 sites elsewhere in the genome of every individual. We compare the sequences of the histone-gene spacers that do and do not contain the insert. The insert is found not to have transposon-like features, and no sequence in the original spacer has been duplicated to flank the insert. There is, however, a hexanucleotide sequence that is repeated three times at one end of the insert, and the element has inserted between direct repeats of 5 bp that were present in the original spacer. One of the copies found outside the histone gene cluster was cloned and sequenced and is compared with the insert. Again, no transposon-like features are evident. Regions flanking the homologous sequence in this clone were used as hybridization probes in whole-genome blots. Results indicate that the 195-bp sequence insert is itself embedded within a larger element that is repeated within the genome. Therefore, only a portion of a larger repetitive sequence has integrated into the histone-gene spacer. The sequence features of the insert, although not typical of mobile elements, may be representative of other illegitimate recombination events.

The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-alpha-trypsin inhibitor also encodes alpha-1-microglobulin (protein HC).

Inter-alpha-trypsin inhibitor (ITI) is a 180 kd serine proteinase inhibitor found in human serum. Treatment of 180 kd ITI with trypsin releases a 30 kd fragment (HI-30) which contains the anti-proteolytic activity of the high molecular weight form. We have isolated a cDNA clone from a human liver library which codes for HI-30, and have determined its DNA sequence. The mRNA not only codes for HI-30 but also another serum protein, alpha-1-microglobulin, which has not been previously associated with ITI or HI-30. The alpha-1-microglobulin sequence is found in the amino-terminus of the protein and is preceded by a signal sequence. HI-30 is found at the carboxy-terminus. The two protein sequences are separated by two arginine residues.

Sequence, organization and expression of late embryonic H3 and H4 histone genes from the sea urchin, Strongylocentrotus purpuratus.

A gene pair coding for histones H3 and H4 expressed during late embryonic development has been cloned from the S. purpuratus genome. The organization of these genes is similar to the divergently transcribed H3-H4 gene pairs of Lytechinus pictus. Whole genome Southern analysis indicates that most late H3 and H4 genes are organized as pairs in both sea urchin genomes. The nucleotide sequences of late and early S. purpuratus H3 and H4 genes differ in the coding regions by 17.0% and 15.7%, respectively. Although there is little match between early and late genes outside the transcription unit, there are short stretches of homology in the spacers 5' to the S. purpuratus early and late H3 genes and the early and late H4 genes. Sequence comparison of the late H3-H4 S. purpuratus pair with two late H3-H4 pairs from L. pictus reveals additional striking homologies in the intergenic spacer. At least four late H3 and two H4 RNAs are distinguished by hybridization to Northern electroblots with probes derived from the cloned gene pair. Although most of these RNAs appeared to accumulate coordinately, with the most dramatic increase occurring between 13 and 17 hours of development, some differences in timing of appearance of different H3 RNA species are observed.

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo.

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.

Evolving sea urchin histone genes--nucleotide polymorphisms in the H4 gene and spacers of Strongylocentrotus purpuratus.

We present a comparison of spacer and coding sequences of histone gene repeats from four Strongylocentrotus purpuratus individuals. Sequences of two previously cloned units (pCO2 and pSp2) were compared with three new histone gene clones, two of them from a single individual. Within a 1.7-kb region, 59 polymorphic sites were found in spacers, in mRNA nontranslated stretches, and at silent sites in codons of the H4 gene. The permitted silent-site changes were as frequent as in any other region studied. The most abundant polymorphisms were single-base substitutions. The ratio of transitions : transversions : single-base-pair insertions/deletions was 3:2:2. A number of larger insertions/deletions were found, as well as differences in the length of (CTA)n and (CT)n runs. Two of the five cloned repeats contained an insertion of a 195-bp element that is also present at many other sites in the genomes of every S. purpuratus individual studied. Pairwise comparisons of the different clones indicate that the variation is not uniformly divergent, but ranges from a difference of 0.34% to 3.0% of all nucleotide sites. A parsimonious tree of ancestry constructed from the pairwise comparisons indicates that recombination between the most distantly related repeats has not occurred in the 1-2 million years necessary for accumulation of the variation. The level of sequence variation found within the S. purpuratus population, for both tandemly repeated and single-copy genes, is 25%-50% of that found between S. purpuratus and S. drobachiensis.

Microtubule protein pools in early development.

Microtubule protein pools have been demonstrated to exist in unfertilized eggs and the early embryonic stages of several organisms. The microtubule pool of the sea urchin embryo is constant in size (about 0.4% of the total embryo protein) throughout early development. Protein withdrawn from this pool for organelle assembly is replaced by new synthesis. Eggs and embryos of Drosophila similarly contain a pool of microtubule proteins (larger than or equal to 0.4% of the total embryo protein, congruent to 3% of the soluble protein), which is constant in size throughout early development. The Drosophila egg microtubule proteins are easily purified by self-assembly in vitro of microtubules, and are similar to microtubule proteins from other organisms in molecular weight and other properties. Synthesis of microtubule proteins in sea urchin embryos is supported by oogenetic mRNA. This appears also to be the case in molluscan (Ilyanassa) embryos. It is not known whether Drosophila embryos synthesize microtubule proteins during the early stages of development.