Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

Dose-dependent schistosome-induced mortality and morbidity risk elevates host reproductive effort

Parasitism changes the host environment and may influence resource allocation between reproductive effort and somatic maintenance. We characterized the impact of dose-dependent schistosome exposure and/or infection establishment on intermediate host survival and reproduction. Four matched groups of Biomphalaria glabrata snails were individually exposed to increasing doses of Schistosoma mansoni parasites, with a fifth control group remaining unexposed. Increased mortality was observed amongst both snails infected and also those snails exposed to the parasite but within which infection did not establish, although only exposed but uninfected snails showed a dose-dependent increase in mortality. Snails also facultatively altered their reproductive output in response to parasite exposure: egg mass production decreased with increasing parasite dose in patently infected snails, whilst, in contrast, exposed but uninfected snails demonstrated a positive association between egg mass production and parasite dose in the post-patent period. These results uniquely suggest an exposure-dose-dependent post-patent fecundity compensation occurring in relation to the risk of future parasite-associated mortality.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.     

Schistosoma mansoni: effect of infection on reproduction and gonadal growth in Biomphalaria glabrata

Sexually mature Biomphalaria glabrata were exposed to 12 miracidia of Schistosoma mansoni, and egg production of snails was monitored over a period of 5 weeks. During the study period, exposed snails grew at approximately the same rate as unexposed controls. Castration, as measured by a reduction in the mean number of eggs laid per snail occurred between 14 and 21 days postexposure (PE). The reduction in fecundity in infected snails coincided with the migration and establishment of daughter sporocysts in the digestive gland and gonad. Enumeration of individual oocytes in longitudinal sections of the ovotestis revealed that uninfected snails contained significantly more oocytes per section than infected snails at 27, 31, and 40 days PE. In addition, the mean area of gonadal sections of control snails increased over the 40-day experimental period, whereas there was no such increase in gonadal area of infected snails. These data suggest that there is an inhibition in gonadal growth in infected snails. When oocyte data were expressed in terms of mean gonadal area, the mean number of oocytes per mm2 of gonad of uninfected and infected snails did not differ significantly over the study period, except at Day 14 PE, when infected snails contained a significantly greater number of oocytes per mm2 of gonad than did uninfected controls. It is hypothesized that daughter sporocysts of S. mansoni are primarily responsible for the inhibition of host reproductive activity, and may be mediating their effects through mechanisms involved in the regulation of gonadal growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of small polydisperse circular DNA from mouse 3T6 cells.

We have isolated, cloned and analyzed small polydisperse circular (spc) DNA from mouse 3T6 cells. The representation of highly repeated mouse genome sequence families in spcDNA has been examined, and the B1 repeat appears overrepresented in spcDNA by two criteria. The majority of spcDNA clones, however, is made out by as yet uncharacterized middle repetitive sequences. We have investigated the increase in the spcDNA population upon cycloheximide treatment of individual sequences, which are found to amplify differentially.     

Comparison of differentially expressed genes in the salivary glands of male ticks,Amblyomma americanum and Dermacentor andersoni

Genes expressed differentially in the salivary glands of unfed and fed male ticks, Amblyomma americanum (L.), were identified, cloned and sequenced, and some were compared with those expressed in the salivary glands of Dermacentor andersoni. Total protein and RNA increased sixfold in the salivary glands of fed male A. americanum, while in fed male D. andersoni salivary glands, RNA increased approximately 3.5 times. Feeding D. andersoni in the presence of females increased total RNA by 25% over those fed in the absence of females. Complementary DNAs were synthesized from RNA obtained from unfed and fed ticks and amplified using RNA arbitrarily primed polymerase chain reaction (RAP-PCR) with three different primers in separate reactions. Differential display showed clear banding differences between the fed and the unfed ticks in A. americanum and D. andersoni. Sixty-one cDNA fragments that appeared to be from differentially expressed genes in A. americanum were isolated, cloned and sequenced. Hybridization reactions with labeled cDNA probes confirmed the differential expression of many of the genes in unfed and fed ticks' salivary glands; however, many of the bands contained more than one fragment and some of the fragments isolated from apparently differential bands were not specific. Sequences for 28 of the cDNA fragments (150-600 nucleotides in length) demonstrated similarity to genes in the databases, but nine of these were similar to sequences of unknown function. Some of the gene fragments identified may be important to tick feeding or tick salivary gland physiology, including a histamine-binding protein, an organic ion transporter, an apoptosis inhibitor, a cathepsin-B-like cysteine protease, proteins involved in gene regulation and several proteins involved in protein synthesis. Cross-hybridization of identified cDNAs from A. americanum with cDNA probes synthesized from D. andersoni total RNA did not show significant similarity between the two species.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Characterization and assessment of an avian repetitive DNA sequence as an icterid phylogenetic marker.

The first tandemly repeated sequence examined in a passerine bird, a 431-bp PstI fragment named pMAT1, has been cloned from the genome of the brown-headed cowbird (Molothrus ater). The sequence represents about 5-10% of the genome (about 4 x 10(5) copies) and yields prominent ethidium bromide stained bands when genomic DNA cut with a variety of restriction enzymes is electrophoresed in agarose gels. A particularly striking ladder of fragments is apparent when the DNA is cut with HinfI, indicative of a tandem arrangement of the monomer. The cloned PstI monomer has been sequenced, revealing no internal repeated structure. There are sequences that hybridize with pMAT1 found in related nine-primaried oscines but not in more distantly related oscines, suboscines, or nonpasserine species. Little sequence similarity to tandemly repeated PstI cut sequences from the merlin (Falco columbarius), saurus crane (Grus antigone), or Puerto Rican parrot (Amazona vittata) or to HinfI digested sequence from the Toulouse goose (Anser anser) was detected. The isolated sequence was used as a probe to examine DNA samples of eight members of the tribe Icterini. This examination revealed phylogenetically informative characters. The repeat contains cutting sites from a number of restriction enzymes, which, if sufficiently polymorphic, would provide new phylogenetic characters. Sequences like these, conserved within a species, but variable between closely related species, may be very useful for phylogenetic studies of closely related taxa.     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.     

一种能表现水稻基因组DNA分化特征的重复DNA顺序

用ＤＮＡ 复性动力学方法克隆到一个水稻中度重复顺序。Ｓｏｕｔｈｅｒｎ 杂交、限制性内切酶分析及序列分析资料表明,该重复顺序在水稻基因组中具有串联重复和散布状态两种存在方式。以该ＤＮＡ 片段作探针,用Ｓｏｕｔｈｅｒｎ 杂交方法分析了多种野生稻种和栽培稻品种的基因组分化特征。某些限制性内切酶消化过的水稻ＤＮＡ,其图谱呈现出多达４０ 条以上的杂交带,包括强杂交带和弱杂交带两种类型。重复实验结果证明,强杂交带表现为ＢＢＣＣ染色体组型特异而弱带则在栽培稻各品种间显示出丰富的多态性,表明该重复顺序片段在水稻理论研究和育种实践中可能具有重要意义     

Biomphalaria tenagophila: genetic variability within intermediate snail hosts susceptible and resistant to Schistosoma mansoni infection

DNA analysis by molecular techniques has significantly expanded the perspectives of the study and understanding of genetic variability in molluscs that are vectors of schistosomiasis. In the present study, the genetic variability of susceptible and resistant B. lenagophila strains to S. mansoni infection was investigated using amplification of their genomic DNA by RAPD-PCR. The products were analyzed by PAGE and stained with silver. The results showed polymorphism between tested strains with four different primers. We found two bands of 1,900 and 3,420 bp that were characteristic of the susceptible strains with primer 2. The primers 9 and 10 identified a single polymorphic band that was also characteristic of (3,136 and 5,041 bp, respectively) susceptible snails. Two polymorphic bands were detected by primer 15: one with 1,800 bp was characteristic of the resistant strain and the other with approximately equal to 1,700 bp in the susceptible one. These results provide additional evidence showing that the RAPD-PCR technique is adequate for the study of polymorphisms in intermediate hosts snails of S. mansoni. The obtained results are expected to expand the knowledge about the genetic variability of the snails and to permit the future identification of genomic sequences specifically related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni.     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).     

Identification of a telomeric DNA sequence in Plasmodium berghei.

A fragment of Plasmodium berghei DNA was cloned using a technique designed to select for telomeric sequences. The cloned fragment recognizes Bal31-sensitive bands in P. berghei genomic digests. It contains at its distal end at least 70 tandem repeats of the heptanucleotide sequence CCCTGAAA. The presence of natural single strand discontinuities in the telomeric regions of P. berghei DNA is demonstrated by the selective incorporation of deoxyribonucleoside triphosphates in the absence of DNase. The number of copies of the cloned sequence present in each genome agrees with an estimate of 6-12 chromosomes per nucleus.     

Isolation of a D-genome specific repeated DNA sequence fromAegilops squarrosa

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa. Nuclear DNA content varies over a range of 10,000 fold among all organisms (Nagl et al., 1983). Among angiosperms, at least a 65-fold range in genome size occurs in diploid species (Sparrow, Price and Underbrink, 1972; Bennett, Smith and Heslop-Harrison, 1982). This DNA variation has been reported within families, genera, and species (Rothfels et al., 1966; Rees and Jones, 1967; Miksche, 1968; Price, Chambers and Bachmann, 1981). Much of the interspecific variation in genome size among angiosperms appears to be due to amplification and/or deletion of DNA within chromosomes. The variation in genome size does not appear to result in changes in the number of coding genes (Nagl et al., 1983). While the number of coding genes, with the exception of rDNA in specific examples, appears to remain constant, the remaining non-coding regions are quite flexible. This non-coding DNA encompasses over 99% of the plant genome and consists of sequences that exist as multiple copies throughout the genome and are identified as repeated DNA sequences (Flavell et al., 1974). Flavell et al. (1974) have reported that increasing genome size in higher plants is associated with increasing repetitive DNA amounts. Subsequent reports have substantiated this correlation (Bachmann and Price, 1977; Narayan, 1982). In various cereals, heterochromatin, which has been demonstrated to be correlated with the location of specific repeated DNA sequences, has been positively correlated with genome size (Bennett, Gustafson and Smith, 1977; Rayburn et al., 1985). Furuta, Nishikawa and Makino (1975) found significant DNA content variation among different accessions ofAegilops squarrosa L. This species contains the D genome, a pivotal genome in several polyploid species and also found in hexaploid wheat (AABBDD). The importance of this genome to the study of bread wheat genomes makes the mechanism(s) of this genomic plasticity of particular interest. In order to determine which sequences are varying, one must first have a way to identify specific types of chromatin and/or DNA. Specific types of chromosome banding such as C- and N-banding have been used to identity types of chromatin in previous studies. C-banding of the D genome results in very lightly staining bands whose pattern is somewhat indistinct. N-banding alternatively has been shown to be useful in identifying certain chromosomes of hexaploid wheat but is limited by the lack of major bands in the D genome (Endo and Gill, 1984). Specific DNA sequences have been isolated fromTriticum aestivum cultivar “Chinese Spring” (hexaploid wheat). However, these sequences are representatives of the A and/or B genomes of hexaploid wheat and are not found in significant quantities in the D genome (Hutchinson and Lonsdale, 1982). Various other repeated DNA sequences have been successfully isolated from rye (Bedbrook et al., 1980) and identified on rye chromosomes (Appels et al., 1981; Jones and Flavell, 1982). Certain of these sequences are found in wheat genomes, but the sequences are representative of only a minor fraction of the D genome (Bedbrook et al., 1980; Rayburn and Gill, 1985). The purpose of this report is to describe three distinct repeated DNA sequences isolated fromA. squarrosa (D genome). Two clones appear to be distributed throughout the total genome, and the third clone is restricted to specific sites along the chromosomes. This latter clone will prove useful in cytologically defining the D genome chromosomes. These sequences appear representative of two types of repeated DNA genome organization: 1) sequences distributed throughout the genome and 2) specific arrays of repeated sequences. The availability of such repeated DNA sequence clones along with the known intraspecific DNA content variation inA. squarrosa will allow the study of genomic plasticity of this species.