Repeat Unit Length Research Articles

Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum.

We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa.

Characterization of a Conserved Extrachromosomal Element Isolated from the Avian Malarial Parasite Plasmodium gallinaceum

We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa.

The multiple codes of nucleotide sequences.

Nucleotide sequences carry genetic information of many different kinds, not just instructions for protein synthesis (triplet code). Several codes of nucleotide sequences are discussed including: (1) the translation framing code, responsible for correct triplet counting by the ribosome during protein synthesis; (2) the chromatin code, which provides instructions on appropriate placement of nucleosomes along the DNA molecules and their spatial arrangement; (3) a putative loop code for single-stranded RNA-protein interactions. The codes are degenerate and corresponding messages are not only interspersed but actually overlap, so that some nucleotides belong to several messages simultaneously. Tandemly repeated sequences frequently considered as functionless "junk" are found to be grouped into certain classes of repeat unit lengths. This indicates some functional involvement of these sequences. A hypothesis is formulated according to which the tandem repeats are given the role of weak enhancer-silencers that modulate, in a copy number-dependent way, the expression of proximal genes. Fast amplification and elimination of the repeats provides an attractive mechanism of species adaptation to a rapidly changing environment.

Regulatory elements of the generic ribosomal gene

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing. The total length of the 45S rDNA repeat unit of S. japonica was 8 995 bp, including 5 420 bp of 18S-5.8S–25S rDNA, and 3 575 bp of IGS (Intergenic Spacer), with the GC content of 51.4 %. The IGS was composed of a 465 bp of 3′-outer transcribed spacer (ETS), an 874 bp 5′-ETS, and a 2 236 bp non-transcribed spacer (NTS), with the GC content of 50.1 %. Fiber-FISH (fiber-fluorescence in situ hybridization) analysis of the distribution of 45S rDNA repeat units on the bacterial artificial chromosome illustrated that each fiber had at least five continuously moniliform hybridization signal points. This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S. japonica. In addition, the successful fiber-FISH analysis of the 45S rDNA on BAC molecule would contribute to the construction of the physical map and map-based cloning of this kelp.

Cross-regulatory interactions among the gap genes of Drosophila

Segmentation of the Drosophila embryo involves expression of several classes of zygotic genes in response to positional information of maternal origin1. Zygotic expression of the gap genes is thought to be required for the subdivision of the embryo into several units of adjacent segments; pair-rule genes establish a repeat unit length of two segments and segment polarity genes specify parts of the single segment2–5. The positional information provided by the three classes of segmentation genes is interpreted for the establishment of segment identity using the homoeotic genes6–10. Molecular probes for several segmentation and homoeotic genes reveal a temporal and spatial pattern of gene expression in the young embryo11–18. Changes in the pattern of expression of these genes in mutant embryos provide evidence for a network of interactions among homoeotic and pair-rule genes19–23 that itself depends on the activity of gap genes24–27. We show here that the spatial limit of gap gene expression depends on bilateral interactions among gap genes.

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket, Acheta domesticus (Orthoptera:Gryllidae).

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.

Length and sequence polymorphisms in the ribosomal gene spacer of the Hawaiian sea urchin, T. gratilla

Blot-hybridization of sea urchin ( Tripneustes gratilla ) genomic DNA with a cloned rDNA probe revealed individual variation in the length of the rDNA repeat unit and also in the non-transcribed spacer sequences. The number of distinct rDNA repeat subclasses distinguishable within any one sea urchin was limited and usually 2 to 3. However, examination of a number of sea urchins indicated a large number of distinct rDNA repeat types in the population as a whole; all of the rDNA repeat types in nine individuals were different. The presence of limited heterogeneity in the rDNA repeats of single individuals, with many different repeat types in the population as a whole, suggests that rDNA variants can be rapidly and selectively propagated within a chromosomal lineage.

Multiplicity of satellite DNA sequences in Drosophila melanogaster.

Three Drosophila melanogaster satellite DNAs (1.672, 1.686, and 1.705 g/ml in CsCl), each containing a simple sequence repeated in tandem, were cloned in pBR322 as small fragments about 500 base pairs long. This precaution minimized deletions, since inserts of the same size as the fragments used for cloning were recovered in a stable form. A homogeneous tandem array of one sequence type usually extended the length of the insert. Eleven distinct repeat sequences were discovered, but only one sequence was predominant in each satellite preparation. The remaining classes were minor in amount. The repeat unit lengths were restricted to 5, 7, or 10 base pairs, with sequences closely related. Each sequence conforms to the expression (RRN)m(RN)n, where R is A or G. The multiplicity of simple repeated sequences revealed despite the small sample size suggests that numerous repeat sequences reside in heterochromatin and that particular rules apply to the structure of the repeating sequence.

Hyper(ADP-ribosyl)ation of histone H1.

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P]ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0). Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.

The organization of a nuclear DNA sequence from a higher plant: Molecular cloning and characterization of soybean ribosomal DNA

The recombinant DNA vector, λ Charon 4A, was used to construct a library of DNA sequences from the genomic DNA of soybean ( Glycine max). To define the organization of ribosomal DNA (rDNA) in the soybean genome, clones containing sequences complementary to both 17S and 25S rRNA have been isolated from this library and used in conjunction with Southern blot hybridization. The rRNA genes are tandemly reiterated with a relatively small unit repeat length of 7.8 kb. There is no heterogeneity in the length of the rDNA repeat units although they display limited differences in either base sequence or pattern of methylation. The cloned rDNA sequences are shown to comprise the entire repeat unit and have been used to obtain a detailed restriction map as well as an approximate transcription map of soybean rRNA genes. The cloning of rDNA from soybean suggests that recombinant DNA techniques can be successfully applied to the genomic DNA of higher plants despite the high degree of methylation exhibited by plant DNA.

Polyimide fibers: Structure and morphology

AbstractPolyimide fibers were prepared by wet spinning of poly(p,p′ ‐diaminodiphenylmethanepyro‐mellitamic acid). Density measurements and x‐ray diffraction studies were carried out to study the structure of the resultant polyimide fibers. Polyamic acid as well as undrawn polyimide fibers were essentially amorphous with two amorphous haloes. Hot drawing of the fibers at 300°C resulted in increase in crystallinity, and a simultaneous decrease in density also took place. X‐ray data revealed that meridional reflections correspond to the repeat unit length in the fiber. Scanning electron micrography studies indicated that polyamic acid fibers prepared by a wet‐spinning technique developed voids during spinning which increased on cyclodehydration to the polyimide state. Hot drawing of fibers resulted in enlargement of these voids. However, a highly fibrillated structure was developed during drawing which could account for the strength of the fibers.

Length and sequence heterogeneity of the histone gene repeat unit of the sea urchin, S. purpuratus

Histone gene repeats in S. purpuratus are shown to be of variable length and sequence. Two recombinant plasmids containing the full-length 6.3 kb histone repeat unit are found to differ in length at two sites in the repeating structure and in the occurrence of two restriction enzyme recognition sites. Variation in repeat length is also demonstrated in the unfractionated DNA of five sea urchins and in a sample of DNA enriched for histone gene sequences by density gradient methods. The repeats in each individual are of a very limited number of major classes, which may differ from one another in overall length or in distribution and presence of particular restriction enzyme sites. Variations are found to occur at many regions of the repeat; some have been mapped specifically to spacer regions. Repeats may differ dramatically from individual to individual since there is no one type of repeat class common to all, although the absolute length differences of the repeats that are found are small.

Arrangement of the rRNA sequences in the rDNA of Lytechinus variegatus

The arrangement of the 26S RNA and 18S RNA sequences of the ribosomal DNA (rDNA) from the sea urchin Lytechinus variegatus was investigated by an electron microscopic analysis of R-loops formed between the ribosomal RNA genes and the mature ribosomal RNAs. Ninety-eight percent of observed molecules contained R-loops clearly seen as a three-stranded complex. The size of DNA complementary to mature cytoplasmic 18S and 26S ribosomal RNA (rRNA) was calculated by measuring the double-strand (ds) and single-strand (ss) part of the R-loops separately. The values for the 18S R-loop are 1.75±0.24 kb1 (ss) and 1.56±0.23 kb (ds). The 26S R-loop is 3.34±0.39 kb (ss) and 3.33±0.33 kb (ds). These measurements agree fairly well with the rRNA sizes measured on denaturing sucrose density gradients: 3.23±0.22 kb for the 26S and 1.93±0.10 kb for 18S. The short spacer between the 18S and 26S R-loops is 1.03±0.24 kb and the longer spacer is 5.36±0.53 kb. In long molecules a repeating pattern was observed. The average length of an rDNA repeat unit is 11.33±0.64 kb when computed using double-strand R-loop measurements and 11.50±0.72 when computed using R-loop single-strand lengths.

Isomorphism in copolyamides containing the p‐phenylene linkage

AbstractSince the observations of Edgar and Hill on polyhexamethylene adipamide/polyhexamethylene terephthalamide copolymers, a linear melting point/composition relationship has become widely accepted as a criterion for isomorphous replacement. In a series of binary copolymers based on homopolymers prepared from hexamethylene diamine and p‐phenylene dipropionic (3P3), 3‐(p‐carboxymethyl) phenyl butyric (2P4), 2‐(p‐carbomethoxy) phenyl propionic (3PO2), hydroquinone diacetic (2OPO2), terephthalic (T), adipic (6), or sebacic (10) acids, only the 6.3P3/6.3PO2 system showed a linear softening point/composition curve, although the differences in repeating unit length were certainly no greater and often much less than with adipic and terephthalic acids. X‐ray examination, however, revealed that all the systems behaved in the same basic manner, the second component dissolving in the lattice of the first until a certain critical concentration was reached when the lattice changed fairly abruptly to that of the second component in which the first component was now dissolved. The changeover point generally, but not always, coincided with the position of a minimum in the softening point/composition curve. Infrared and density measurements did not show any reduction in crystallinity at this point. These copolyamides, thus, afford no exception to the assumption that close similarity in length of repeating units is a necessary, but perhaps not the only, prerequisite for the occurrence of segmental change. The form of the softening point/composition curve is, therefore, not a reliable criterion for isomorphous replacement and it seems likely that this conclusion applies equally to the melting point/composition curve.

The infra-red spectra and molecular configurations of polyamides

The infra-red spectra of a wide variety of polyamides of diamine-diacid and ω-amino acid type are reviewed. The amide-amide dipole interaction is the dominant factor controlling the packing and configurations of the molecules. Complete association occurs in all polymers and copolymers. This imposes a distortion on the hydrocarbon segments out of the planar zig-zag configuration in the copolymers with differing repeat unit lengths, and in the diamine-diacid polymers with odd numbers of CH 2 groups in either or both sections of the repeat unit.