RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y

Summary Probes of uncloned Bkm satellite DNA and a Drosophila clone 2(8), consisting mainly of GATA repeasts related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistint series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ.     

Campoletis sonorensis Endoparasitic Wasps Contain Forms of C. sonorensis Virus DNA Suggestive of Integrated and Extrachromosomal Polydnavirus DNAs

Campoletis sonorensis virus (CsV) (Polydnaviridae) previously was detected only in the calyx epithelial cells and lumen of the oviducts from female C. sonorensis (Ichneumonidae) endoparasitic wasps (Norton et al., Cell Tissue Res. 162:195-208, 1975). Using dot-blot hybridizations, we detected low amounts of CsV DNA in male and female wasp head and thorax tissues and in male abdominal tissues. Low amounts of extrachromosomal viral DNA were detected in Southern blots of undigested male wasp DNA and in male DNA purified by isopycnic centrifugation. High-molecular-weight male wasp DNA digested with any of several restriction endonucleases and hybridized with cloned viral DNAs from CsV superhelices B and Q under stringent conditions contained CsV-specific DNA fragments that differed significantly in size and number from the hybridizing fragments detected in comparably digested viral DNA. Identical offsize restriction fragments were detected in digested female head and thorax DNA. These data suggest that at least CsV DNAs B and Q are integrated in C. sonorensis cellular DNA and that the virus may be transmitted through the germline.     

Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Chemical Characterization of Leaves,Male and Female Flowers from Spontaneous Cannabis (Cannabis sativa L.) Growing in Hungary

Spontaneous forms of hemp (Cannabis sativa L., often reported as Cannabis sativa var. spontanea Vavilov ) with a low content of psychoactive cannabinoids can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this article, we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analyzed using GC/MS, NMR and LC‐DAD‐MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists, (E)‐caryophyllene (28.3 %) and cannabidiol (CBD; 24.9 %), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC/MS allowed to quantify cannabidiol (CBD) and cannabidiolic acid (CBD?A) in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9 %, and 0.2, 0.3 and 0.4 %, respectively. Flavonoids of this spontaneous form of hemp were formed by C‐glycosides and glucuronic acids of kaempferol and apigenin with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, spontaneous C. sativa may represent an important source of CB₂ agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

Male-specific DNA sequences in pigs

One hundred primers (Operon kits OPAA, OPAO, OPAV, OPC, and OPE series) were used for random amplified polymorphic DNA (RAPD) fingerprinting to determine male-specific fragments. Seventy-four percent of the primers yielded Yorkshire polymorphic fragments. One of these primers, OPAV-18, produced a novel 1098-bp DNA fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing. Two primers (5'-TTGCTCACGG TAGATAACAA GAGAG-3' and 5'-TTGCTCACGG ACCAGGTAGG GAATG-3') were designed according to the cloned male-specific sequence to amplify the male-specific band using polymerase chain reaction (PCR) for pig sexing. Sex-specific bands in the PCR gel products were represented in males but none were found in females when Yorkshire, Duroc, and Landrace genomic DNA samples were amplified with these two primers by PCR. The PCR products in the gel were transferred to nylon membranes and hybridized with a 32P-dCTP labeled probe of the cloned male-specific DNA fragment. There was a clear hybridization signal in samples from all of the male pigs, but not from those of female pigs. Male and female genomic DNA samples from these pigs were spotted onto nylon membranes and hybridized with the male-specific probe. The probe hybridized strongly to males only. A high degree of sequence homology was found among the novel male-specific DNA sequences in Yorkshire, Duroc and Landrace. The sex of these three breeds of pigs could be easily and effectively determined using these two primers.     

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.     

银杏雌雄基因组DNA间的差异性分析

本研究应用RAPD技术,应用300个10bp随机单引物及200对随机双引物组合,检测了雌雄异株银杏基因组DNA的多态性。结果表明:雌雄基因组间具有极高的相似性,在检测到的3450个标记中,仅获得1个与银杏雄性基因组相关的RAPD标记。以该标记为探针,与雌雄银杏基因组DNA的Southern杂交分析,其杂交信号在两性之间表现为限制性片段长度多态性,该结果为寻找银杏早期性别鉴定的探针以及在细胞和分子水平进一步研究其性别问题奠定了基础。     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

Gynodioecy in Cirsium chikushiense Koidz. (Compositae)

A study of the morphology and function of flowers in Cirsiumchikushiense revealed that the species was distinctly gynodioecious.Self-incompatible hermaphrodite florets produced both seedsand pollen grains, while female ones produced seeds but no pollengrains at all. The degenerated stamens of females were not onlysmaller but also sometimes occurred at a lower position insidethe floral tube than in hermaphrodites. The stigmata of femalesoften developed more fully than those of hermaphrodites. Thefrequencies of female plants in natural populations varied from15·5 to 50%. Almost all the pollinators stayed on bothfemale and hermaphrodite heads only to collect nectar. The femaleplants of this species may be more specialized in their genderby saving the cost of not only pollen grains but also stamens,and may be maintained by sufficient pollinators in natural populations.This gynodioecy may provide an example of nuclear-cytoplasmicmale sterility.Copyright 1994, 1999 Academic Press Sex expression, gynodioecy, Cirsium chikushiense, Compositae, male sterility, degenerated stamen, female frequency     

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.     

The role of roots in sex expression in hemp plants

When the shoots of young hemp (Cannabis sativa L.) plants were cut off the roots, cultured as cuttings, and regenerating (adventitious) roots were removed as soon as appearing, ca. 80–90% of the plants became male (had staminate flowers) whereas if the roots were allowed to develop a similar percentage became female (pistillate flowers). Treatment of de-rooted cuttings with 6-benzylaminopurine (15 mg/l) restored the percent of female plants to ca. 80. It is suggested that the root system plays an essential role in sex expression in hemp and that this role is related to cytokinin synthesis in the root.     

Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes

Abstract Various arbitrary primers as well as pUC18/19 'reverse' sequencing primers were used for random amplified polymorphic DNA assays. Use of a modified reverse primer led to amplification of one major approx. 1100-bp band from the chromosomal DNA of all actinomycetes tested; however, the band was not found when DNAs from other bacteria were used in comparable experiments. Hybridization experiments showed that these bands all contained similar genomic regions. Subsequent sequencing of four of these fragments showed they each contained the sequence of the 3' end of the 23S rRNA gene, the intergenic region and the start of the 5S rRNA gene.     

Sex-ratio differences in Mnium hornum Hedw. and M. undulatum Sw. in relation to spore germination and vegetative regeneration

As Mnium undulatum was shown to be homosporous, it was concludedthat neither male nor female derived an advantage from sporesize that might be related to the observed excess of femaleplants. The rate of germination was greater at 20 °C thanat 10 °C in M. hornum and M. undulatum, and was also reducedin short days (7.25 hours) at both temperatures. Spores of M.undulatum germinated more slowly than those of M. hornum undereach of the environmental regimes used. Isolated spores of M.undulatum showed a ratio of 1: 4.1 compared with 1: 0.89 inM. hornum. The excess of female plants of M. undulatum thathad been established by the end of germination, was maintainedamongst the first protonemal buds produced (1: 3.5), whereasan excess of male M. hornum was observed in the first protonemalbuds (1: 0.45). Frost reduced the rate of germination in M.undulatum, but unlike desiccation did not affect the final percentage.Male and female were amongst the spores which survived desiccationat 10 °C. Regeneration of detached leaves occurred more rapidly in M.undulatum than in M. hornum, and no difference between maleand female was detected. It was found that frost prior to orduring regeneration did not produce long-term harmful effectsin M. undulatum. None of the young male gametophytes producedby regeneration from leaves survived desiccation, compared with77 per cent of similarly produced female gametophytes.     

Identification of fast and slow growing rhizobia nodulating soybean (Glycine max [L.] Merr) by a multiplex PCR reaction

Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes nolBT of Sinorhizobium fredii and the other one homologous to RSalpha, an insertion-like sequence present in Bradyrhizobium japonicum, were generated by polymerase chain reaction (PCR) with two pairs of primers. The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present. The amplification of the 900-bp RSalpha fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp nolBT fragment. By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between S. fredii, Bradyrhizobium japonicum and Sinorhizobium xinjiangensis.     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。