Transposition of mobile genetic elements in interspecific hybrids of Drosophila

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using ³²P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions (jumping) of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with synthetic karyotoypes characterized by different combinations of D. virilis homologous chromosomes and hybrid chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition (jumping) took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such synthetic stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Replication analysis of plasmid DNAs injected into Drosophila embryos

From a shotgun collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and nonmethylation. within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.by J.A. Huberman     

Glycoconjugate Expression During Early Mouse Oculogenesis

The carbohydrate side-chain of glycoconjugates can show a developmentally regulated expression pattern. In order to analyse these changes during the development of the eye, 13 lectins were used to reveal glycoconjugates histochemically in 8.5- to 14-day- old mouse embryos. During this period, eyes develop from the most immature vesiculation of the neural plate neuroepithelium into a primitive stage with all structures present, such as pigment epithelium, not yet differentiated neuroretina and lens. A striking diversity of carbohydrate side-chain expression was observed in the preocular somatoectoderm and neural plate of 8.5- day-old embryos, as indicated by the binding of nine different lectins. Binding sites at the apical poles of neuroepithelium of five of these lectins (PNA, LCA, SBA, LPA and GSA-II) disappeared completely during further development. The binding sites of four other lectins, WGA, MPA, Con A and BPA, remained expressed during the course of development, being indicative for the carbohydrate side-chains -GlcNAc(1-4)Gluc, -Gal(1-3)GalNAc, -D-Man/-D-Gluc and -GalNAc. In contrast, binding sites for GSA-I, RCA-I (-D-Gal), UEA-I (-l- Fuc) and DBA (-GalNAc(1-3)GalNAc) were not present at any developmental stage. The time point of gross changes of lectin binding sites correlates well with the period of neural tube formation. During later development from neuroectoderm to the ocular pigment epithelium, a sharp reduction in all lectin binding sites at the apical cell poles, except for WGA and MPA, was observed. WGA binding sites were present until embryonic day 10, while those for MPA were present until day 9. At the basal cell poles of the pigment epithelium, all lectin binding sites except for WGA were lost after e mbryonic day 11.5. These results indicate that there are sophisticated kinetics of glycoconjugate expression during the course of early embryonic development of ectoderm into its descendent tissues.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Developmental asynchrony caused by steep temperature gradients does not impair pattern formation in the wasp,Pimpla turionellae L.

Generating developmental gradients by temperature gradients established within a developing organism is an easy, non-invasive technique to study physiological interdependencies between locally separated subsystems. A linear temperature gradient of about 10° C/mm was maintained up to 5 h in either direction along the long axis of a long-germ-type hymenopteran egg, which was simultaneously filmed by the 16 mm timelapse technique. The result was a dramatic desynchronization of development, which between the egg poles could reach up to 9.3 h relative to normal development. Within the same egg, up to seven mitotic waves (i.e. eight different nuclear generations) were observed at the same time, and the subsequent cellularization process was extremely asynchronous. The initial regions of the mitotic waves, the fountain flow of the ooplasm, and the gastrulation process were shifted towards the egg pole kept at higher temperatures. Developmental processes occurring successively in normal development now took place simultaneously, with either direction of the temperature gradient. For instance, while gastrulation had started in the warm region, midblastula transition and cellularization were in progress in the middle of the egg, and intravitelline nuclear multiplication occurred at the cold pole, by rapid and still biphasic cell cycles. In some respects, development resembled that of a short-germ-type insect egg. Nevertheless, the developmental processes were resynchronized after the temperature gradient was switched off. Surprisingly, the extreme desynchronization during early development did not affect the segment pattern of the resulting embryos. The technique of inducing well-defined developmental asynchronies might be applied in Drosophila to analyse the subtle interplay between maternal and zygotic gene activities described in this species.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Host-dependent modification of bacteriophage T7 and SAMase-negative T3 derivatives affecting their adsorption ability

Summary When passaging phage T7 and SAMase-negative T3 mutants betweenE. coli strains with identical (EcoB) or without (EcoO) DNA host specificity, phenotypically a host-controlled modification and restriction is observed. This phenomenon is not due to classical modification and restriction of the bacteriophage DNA but depends on the reversibly altered adsorption capacity of the phages on the different host strains.     

Evidence against DNA polymerase β as a candidate gene for Werner syndrome

Werner syndrome (WS) is a rare autosomal recessive disorder of humans characterized by the premature onset and accelerated rate of development of several major age-related disorders. An aberration in DNA replication or repair is suggested by the evidence of genome instability. Since the structural gene for DNA polymerase maps within the region of the WS mutation on the short arm of chromosome 8 and is involved in both DNA repair and DNA replication, we evaluated its candidacy as the WS gene. Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

Induction of embryogenicTriticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on induction of embryogenic calli from immature embryos of a responsiveTriticum aestivum L. genotype (PCYT 10). Effects were quatified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M signficantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mgl^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D inT. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3359.     

Classification and intragenic position of mutations in the beta-galactosidase gene of Escherichia coli

Summary Following treatment with N-methyl-N-nitro-N-nitrosoguanidine, 1,257 mutants of Escherichia coli K12 were isolated on lactose-tetrazolium medium. Of these mutants, 345 were lactose-negative and lacked appreciable -galactosidase activity. About half of these enzyme-deficient mutants had lost the whole lactose operon; the remainder (174) were point mutations within the -galactosidase gene. With the exception of 42 which could not be classified, the mutations were identified either as chain-terminating (UAG 57, UAA 6, UGA 60) or missense (9). There were no mutations of the reading frame and no short deletions. The unclassifiable mutants do not form crossreacting protein and are probably a type of chain-terminating mutant.Each of the point mutations was assigned by sexduction to one of 36 deletion regions. The map distribution shows a deficiency of chain-terminating mutants in the operator-distal part of the gene and a localization of missense mutations in regions corresponding to the position of the apparent substrate-binding sites of the enzyme.     

“Hidden” popular illnesses in primary care: Residents' recognition and clinical implications

This study documents that ethnomedical beliefs and practices play an important role in primary care in a southern community. Thirty-three of 73 patients from a rural Appalachian area coming to a university primary care internal medicine practice presented 54 ethnomedical complaints such as high blood (24.1%), Weak 'n dizzy (22.2%), nerves (16.7%), sugar (5.6%) and fallin' out (3.7%). Thirty-three patients had both biomedical and ethnomedical complaints, 40 patients had biomedical complaints without ethnomedical complaints and no patients presented with ethnomedical complaints alone. Over two-thirds of all patients consulted non-medical personnel for their complaints, mostly family and friends, and 70 percent self-treated prior to clinic consultation. Patients presenting with ethnomedical complaints when compared with those presenting with biomedical complaints sought advice of non-physicians significantly more often (p < 0.02); no statistical difference, however, was found in their self-treatment practices. Ninety-two of 130 biomedical complaints were recorded by the patient's physician but none of the 54 ethnomedical complaints were formally recorded (p < 0.001). The high incidence of ethnomedical complaints in this population and the failure of physicians to recognize these complaints demand that primary care medicine residents be taught improved history-taking skills and the essentials of ethnomedical illnesses if they are to provide culturally-sensitive patient care. [ethnomedicine; physician education; Southern black and white Appalachian folk medicine; culturally-appropriate primary care.]     

Replication forks are not found in a Drosophila minichromosome demonstrating a gradient of polytenization

Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The underreplication model of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.by W. Hennig     

Über quantitative Veränderungen „gomoripositiver“ Substanzen in Infundibulum und Hypophysenhinterlappen der Ratte nach beidseitiger Adrenalektomie

Zusammenfassung Bei der normalen weißen Ratte verhält sich die Zona externa des Infundibulum im Gegensatz zur Zona interna infundibuligomorinegativ.Am 7. Tag nach beidseitiger Adrenalektomie treten in der Zona externa gomoripositive Substanzen auf, deren Menge über den 14. Tag nach der Operation hinaus zunimmt.Im supraoptico-hypophysären System ergibt die quantitative Messung eine Entleerung des neurosekretorischen Materials am 3. Tage nach beidseitiger Adrenalektomie. Im Verlauf von 14 Tagen nach dem operativen Eingriff füllt sich das neurosekretorische System wieder bis zu den beim Normaltier ermittelten Werten auf.Die gomoripositiven Granula in der Zona externa inf. sind möglicherweise das morphologische Äquivalent eines Corticotropin-releasing factors.

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On quantitative changes of Gomori-positive substances in the median eminence and neural lobe of the rat hypophysis after bilateral adrenalectomy

Summary In the normal albino rat, the external layer of the median eminence shows a Gomori-negative histochemical reaction as opposed to the internal layer.On the 7th day after bilateral adrenalectomy, Gomori-positive substances appear in the external layer, their amount increasing up to and beyond the 14th day p.o.In the supraoptico-hypophyseal system, quantitative measurements show a depletion of the neurosecretory substance on the 3rd day following bilateral adrenalectomy. This loss of neurosecretory material is restored during the course of 14 days p.o., the amount then corresponding to that found in the normal rat.It is conceivable that the Gomori-positive granules in the outer layer of the median eminence are the morphological equivalent of a CRF.

     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.