The relationship of the "sphere chromatophile" to the fate of displaced histones following histone transition in rat spermiogenesis.

Cytochemical, radioautographic, and microspectrophotometric studies bearing on the relationship of histone transition to the origin and development of the protein and RNA components of the "sphère chromatophile" in the developing spermatogenic cells of the albino rat are presented. These studies show that the sphère chromatophile has many features in common with somatic nuclei: it contains histonelike basic proteins rich in lysine, with lesser amounts of arginine. No evidence is found for the presence of a protamine in this granule. The sphère chromatophile is rich in RNA, but contains no DNA. The failure of a positive reaction with basic protein stains, unless the RNA is first removed, indicates either a chemical bonding or a very close association between the RNA and basic protein. The basic protein and RNA components of the sphère chromatophile appear to have different origins in the cell. A sequence of stages in the development of the lysine-rich basic protein component of this structure commences with the appearance of tiny grains in those spermatid nuclei which are beginning to replace lysine-rich histones with arginine-rich histones. Subsequently, similar-staining cytoplasmic grains appear, which coalesce to form the sphère chromatophile in the cytoplasm. Labeling studies show that the basic protein component is synthesized at about the time of the last premeiotic DNA (and histone) synthesis. The results of the microspectrophotometric measurements support the idea that the basic protein lost from the spermatid nucleus is the source of the basic protein in the sphère chromatophile.

A kinetic study of DNA and basic protein metabolism during spermatogenesis in the sand crab, Emerita analoga.

Cytochemical and radioautographic techniques define and confirm a staging scheme for developing spermatids of the decapod crab, Emerita analoga. Quantitative photometric data demonstrate that developing spermatids lose a significant proportion of their nuclear proteins, as evidenced by diminishing binding of fluorodinitrobenzene. Photometric results also show that much (but not all) of the spermatid nuclear protein loss is in somatic-type histone, as evidenced by a dramatic fall in the histone/DNA ratio of these cells during a period in which nuclear DNA content remains constant. By the end of spermiogenesis, the sperm nuclear histone and protamine content is approximately zero, whereas some nonbasic protein persists. Loss of spermatid nuclear somatic-type histone is not accompanied by synthesis of gamete-type histone (e.g. protamine or arginine-rich histone), showing that the processes of displacement and synthesis of nuclear basic proteins during histone transition are not subject to obligatory coupling. Labeling studies suggest that nonbasic acrosomal proteins (presumably partly enzymes) are synthesized in the cytoplasm, after which they move into the acrosome. Stainable basic proteins accumulate in the acrosome during precisely the period of nuclear somatic histone loss, suggesting nuclear-cytoplasmic transfer.

Evidence of paleozoic chromosomes from lycopod microgametophytes.

A Pennsylvanian arborescent lycopod cone, Lepidostrobus schopfii, has microspores that have been found to have intracellular features that are interpreted as nuclei and mitotic chromosomes. The cellularized gametophytes conform to the early stages of growth that occur in modern Selaginella microgametophytes. Since the megagametophyte of L. schopfii is similar in development to extant species of Isoetes, the fossil now is known to have portions of its life cycle in common with both Selaginella and Isoetes.

Instrumentation needs of research universities.

This article assesses the status of scientific instruments in major research universities and identifies factors that facilitate or impede their development, acquisition, use, and maintenance. Sixteen universities, six national and government laboratories, and nine commercial laboratories were visited; over 700 individuals were interviewed. Data on instrument acquisition and age were collected. Instrumentation was examined in physics, chemistry, biological sciences, earth sciences, and electrical engineering. The study found that the quality of university instrumentation has seriously deteriorated, due principally to a relative decrease in instrumentation funding, inflexibility within the project grant system, and insufficient support for maintenance.

Ribosomal RNA sequence conservation and gene number in the larval brine shrimp.

The haploid genome size of Artemia is determined to be about 0.9 X 10(12), as evidenced both by Feulgen microspectrophotometry of individual diploid class nuclei, which are but one of five polyploid classes present within the larvae, and by analysis of the reassociation kinetics of the isolated single copy DNA component. Polysomes isolated from 24-h incubation stage larvae contain an average of 10 ribosomes per messenger RNA molecule. Their rRNAs are found to have sedimentation coefficients of 18 S and 26 S, corresponding to molecular weights of 0.70 X 10(6) and 1.40 X 10(6), respectively, as determined by polyacrylamide electrophoresis and also by sucrose density centrifugation. Denaturation in glyoxal followed by agarose gel electrophoresis shows that unlike deuterostome rRNAs, Artemia 26 S rRNA contains a cryptic nick about midway in the molecule, which is not found in the 18 S molecule. Isolated rRNAs were labelled in vitro with 125I and hybridized with filter-immobilized DNA to saturation, which occurred at 0.051% for Xenopus, and at 0.074% for Artemia. From these results, it is calculated that in the haploid Artemia genome there are about 320 copies of the (18 S + 26 S) ribosomal RNA genes. Reciprocal heterologous hybridizations between these two species show that they share about 30% homology between their rDNA coding sequences.

Mitochondrial RNA editing of cytochrome c oxidase subunit II (coxII) in the primitive vascular plant Psilotum nudum.

A 634-nucleotide tract, including primers, was amplified via the polymerase chain reaction within a mitochondrial coxII gene of the primitive vascular plant Psilotum nudum and sequenced. Alignment with homologous coxII gene sequences from diverse plant species having known RNA editing sites which restore amino acid sequence consensus was used to infer eight sites of C-to-U transitions in Psilotum. In every case, the predicted editing event would confer the selective advantage of conserving the amino acid residue at a site where amino acid sequence divergence has not been observed in other plant species. The plant mitochondrial editing machinery is shown for the first time to extend to one of the deepest branches of vascular plant phylogeny.

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp.

A library of genomic DNA from the brine shrimp, Artemia, has been constructed with the Charon 4A phage vector, utilizing EcoRI passenger fragments. Screening this library with purified Xenopus laevis cloned rDNA genes has resulted in the identification and plaque purification of a recombinant containing a complete Artemia (18 S + 26 S) rDNA repeat unit. A physical map derived from the analysis of restriction endonuclease digests of the repeat unit, which measures 13.9 kilobase pairs, is similar to the map derived from genomic DNA. In common with several other species, the 26 S rRNA gene terminates with a HindIII recognition site.

Universality of mitochondrial RNA editing in cytochrome-c oxidase subunit I (coxI) among the land plants.

Plant mitochondrial pre-mRNAs often undergo C-to-U conversions, a phenomenon termed RNA editing. The molecular source of specificity and phylogenetic depth of the editing machinery remain to be determined. We amplified coxI gene fragments via the polymerase chain reaction from a diversity of taxa within the land plants, and sequenced each. Alignment and comparison of 25 homologous coxI gene sequences with those from plant species having known RNA editing sites which restore amino acid sequence consensus was used to infer sites of C-to-U conversions. Our results, derived using the comparative approach, imply that the plant mitochondrial editing machinery extends throughout vascular plant phylogeny, and also that this phenomenon is present in every major branch of the (non-vascular) Bryophyta: liverworts (Hepaticae), hornworts (Anthocerotae), and mosses (Musci). These results have important consequences for our thoughts on the evolutionary history of the plant RNA editing process, as they imply that editing is older than was previously believed.

RNA editing in Drosophila 4f-rnp gene nuclear transcripts by multiple A-to-G conversions.

Pre-mRNA editing results in production of transcripts having nucleotide sequences differing from that of the DNA template. We describe the first example of RNA editing in the fruitfly Drosophila melanogaster. This editing occurs in an alternatively spliced adult head transcript arising from the single-copy nuclear 4f-rnp gene via numerous A to G conversions. Editing sites were identified from comparisons of the genomic DNA sequence with that of corresponding cDNAs prepared from various developmental stages. We show that only the non-edited sequence is present in wild-type fly chromosomes, and have conducted a genetic rescue experiment that suggests that the edited cDNA is expressed in a protein that partially complements a lethal 4f-rnp mutation. The extensive editing observed is predicted to significantly alter the amino acid sequence of the encoded protein, and thus could provide a novel mechanism for the modulation of gene expression in Drosophila. The true significance of our discovery is that it was made in Drosophila, and the advantages of using Drosophila as a genetic model to further study RNA editing in the 4f-rnp gene are discussed.

Interspersion of histone and 5S RNA genes in Artemia.

Four recombinant lambda phage containing histone genes were selected from a library of Artemia genomic DNA fragments. The histone gene organization of Artemia resembles that of other invertebrates in that all five genes are clustered and repeated in tandem with approximate repeat lengths of 8.5 kb and 9.3 kb. Each recombinant lambda phage isolate hybridizes with five histone mRNAs and unexpectedly also with 5S ribosomal RNA. Hybridization kinetics have shown the number of histone genes to be about 95-100 copies per haploid genome. An identical number of copies was determined for a hybridization probe containing the 5S gene but no histone genes. We have not found any evidence for a separate set of repeated 5S genes outside this histone + 5S block.

RNA editing and alternative splicing generate mRNA transcript diversity from the Drosophila 4f-rnp locus.

Extensive sequencing of genomic 4f-rnp and 15 cDNA clones isolated from libraries of 0-4-h embryos, pupae, and adult heads from Drosophila melanogaster has enabled identification of factors resulting in transcript sequence diversity. The 4f-rnp gene contains eight small introns. Among non-edited cDNAs, one transcript class potentially encodes a 943-amino-acid protein, within which several motifs are predicted, including a single C-terminal RNA-binding domain. Intron 5 is retained in all cDNAs examined except for a pupal cDNA, where it is excised. This potentially introduces an in-frame stop codon and predicts a truncated protein of 639 amino acids. One adult head transcript class is edited, some 31% of As being converted to Gs exclusive of edits within introns. An edit site in intron 4 changes a canonical 3'-terminal AG to GG, which interferes with splicing and is predicted to introduce a stop codon. A potential editing substrate recognition element in 4f-rnp contains the weak consensus sequence: 5'-G-G-G-N-A-A-G-3', which may interact with double-stranded RNA adenosine deaminase following pairing of 4f-rnp mRNA with an antisense transcript. It is possible that extensive editing in 4f-rnp destabilizes transcripts and thus provides a novel mechanism for post-transcriptional control of gene expression, although resolution of this point will require study of additional edited transcripts.

The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion.

Levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) protein and neurotrophin receptor mRNA in adult sympathetic neurons were investigated following surgical removal of preganglionic input and/or in vivo administration of NGF. Expression of trkC and p75, but not trkA, was significantly decreased following a 3-week deafferentation of the superior cervical ganglion (SCG). Protein levels of NGF and NT-3 in the SCG were unchanged by deafferentation. A 2-week intracerebroventricular infusion of NGF without deafferentation resulted in enhanced mRNA levels of trkA, trkC, and p75 as well as significantly increased NGF and NT-3 protein in the SCG. When NGF infusion followed deafferentation, both trkA and p75 showed significant increases while trkC levels were similar to control values. NGF protein was not increased in the SCG when deafferentation preceded exogenous NGF, yet NT-3 was elevated and levels were similar to cases receiving NGF infusion only. These results support a role for preganglionic input in trkC and p75 expression in adult sympathetic neurons. The increased levels of NT-3 protein and trkC gene expression observed following NGF infusion suggest that NGF influences NT-3 regulation in adult sympathetic neurons. In addition, the present findings provide evidence that, when preganglionic input is removed prior to the NGF infusion, NT-3 effectively competes with NGF for trkA binding. Taken together, we propose that NT-3 may play a role in the robust sprouting of sympathetic cerebrovascular axons previously observed following NGF administration, particularly when deafferentation precedes the NGF infusion period.

DNA reassociation kinetics and chromosome structure in the crabs Cancer borealis and Libinia emarginata.

DNA reassociation kinetics have been partly elucidated for the higher crabs C. borealis and L. emarginata, using calf thymus DNA as a standard. These crabs contain no detectable repeated DNA in the approximate multiplicity frequency range 2-100 copies, which is unusual for invertebrate DNAs. Each species contains a component renaturing at an intermediate rate, and also a very rapidly renaturing fraction. The very rapidly renaturing fraction is considerably larger than the cesium chloride-resolvable satellites of each species. The fraction reassociating at an intermediate rate includes sequences with a reiteration frequency of up to 9.0 X 10(4) copies. This is unusually high for invertebrate DNAs. The nearly exact correlation between kinetic complexity and independently determined haploid genome size leads to the conclusion that the most slowly renaturing sequences of both crab species are present only once per haploid genome. Therefore the chromatids of these species are uninemic structures, and there has been no detectable occurrence of polyploid speciation in the recent evolutionary history of either species.

Conservation of repeated DNA base sequences in Crustacea: a molecular approach to decapod phylogeny.

Analysis of data obtained from molecular hybridization of 3H-labeled repetitious DNA has been utilized to reconstruct the broad outlines of phylogenetic relationships among decapod Crustacea. This molecular reconstruction agrees reasonably well with the paleontological record, and with other schemes obtained by comparative morphological and serological approaches. Preliminary evidence is in line with the hypothesis that continuous addition of new repeated sequence families to the genome over long periods of time may in part account for the correlation observed between percent repetitious DNA hybridized and divergence time. It is tentatively concluded that a core of DNA base sequence homology has been highly conserved throughout the evolution of the Crustacea. Demonstration of inter-species sequence homology has important implications to models which relegate a genetic regulatory function to repeated DNAs.

Restriction endonuclease mapping of ribosomal RNA genes: sequence divergence and the origin of the tetraploid treefrog Hyla versicolor.

Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the "western" diploid population and about 50% resembling the "eastern" population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5' terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.

Physical and biochemical characterization of the cloned LYS5 gene required for alpha-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae.

The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.

The Peperomia mitochondrial coxI group I intron: timing of horizontal transfer and subsequent evolution of the intron.

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out.

In vivo transcription from multiple spacer rRNA gene promoters during early development and evolution of the intergenic spacer in the brine shrimp Artemia.

The control of ribosomal RNA (rRNA) gene expression during development can be productively studied by examination of the relationship between promoter structure and function as well as the processing of primary transcripts. Toward this end total cell RNA was extracted from embryos at various stages and probed with cloned rRNA genes using the "dot blot" method. This exercise showed that rRNA gene expression is a stage-specific process and is thus under developmental control. S1 nuclease protection experiments localized fourteen different upstream DNA sites encoding 5'-termini of pre-rRNAs during this synthetic phase of development. There is no indication of any spacer fail-safe terminator function. The S1 approach contributed to the sequencing of several of the sites. Comparative sequence alignments reveal short conserved regions in DNAs corresponding to these sites, which are shown to fall into two structural classes. Sites 3, 4, 6 and 9 are proposed to function in transcription initiation and are found to have the consensus sequence 5'...T-A-T-A-T-Pu-Pu-Pu-G-Pu-Pu-G-T-C-A 3'. Sites 1, 2, 5 and 8 which are proposed to function in 5'-processing have the consensus sequence; 5'...Pu-G-T-Pu-T-T-G 3'. These short sequence conserved regions are hypothesized to serve as recognition signals for proteins within the rDNA transcription initiation complex and for 5'-processing enzymes, respectively. Sequencing of the intergenic spacer region from which a model for spacer evolution is derived shows that tandem ca 600 bp subrepeats explain much of the multiplicity observed within control sites.