The association of the human ε-globin gene with the nuclear matrix: a reconsideration

The association of the human -globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human -globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G/A-globin gene domain, the 16 kb /-globin gene domain and the 10 kb -globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association.Summarizing our results we could say that a region starting 5.7 kb 5 to the human -globin gene and ending 4 kb 3 to the human -globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.     

Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana

The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector gtB. Three different recombinants (gtRa1, gtRa23 and gtRa5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The gt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.     

β-Globin gene polymorphism in the Saudi Arab population

Summary DNA mapping with the restriction endonucleases, Hpa I and Mst II, has been used to investigate -globin gene polymorphism in the Saudi Arab population. Using Hpa I digestion, 13.0kb and 7.6kb fragments were found in association with the ^A and ^S genes. The frequency of the polymorphic forms in two regions investigated vary significantly. In Al-Hafouf and the surrounding villages, situated in the Eastern Province of Saudi Arabia, the frequencies of association of the ^S gene with the Hpa I 7.6kb, 7.0kb, and 13.0kb fragments were 0.866, 0.043, and 0.071, respectively. The frequency of association of ^A with the 7.6kb and 13.0kb fragments resulting from the Hpa I digestion were 0.875 and 0.125. In Khaiber, Tehamat-Aseer, and surrounding villages, in the Western Province, the frequency of association of ^S with 7.6kb, 7.0kb, and 13.0kb fragments were 0.836, 0.027, and 0.0136, respectively, while that of ^S was 0.250 and 0.750 with 7.0kb and 13.0kb Hpa I fragments, respectively. Using Mst II digestion, ^A was found to be linked to a 1.15kb fragment, while ^s was linked to a 1.35kb fragment. The normal (Hb AA), heterozygotes (Hb AS), and homozygotes (Hb SS) gave 1.15, 1.15/1.35, and 1.35kb fragments, respectively. The results of this study show extensive polymorphism at the Hpa I restriction site of the ^A and ^S globin genes with the different polymorphic forms existing at a variable frequency in different regions of Saudi Arabia.     

The supramolecular organization of red algal cell membranes and their participation in the biosynthesis and secretion of extracellular polysaccharides: a review

Summary The relationship between the supramolecular organization of red algal cell membranes and the biosynthesis and secretion of the cell wall skeletal and matrix polysaccharides is reviewed. Freeze-fracture studies have revealed that organized macromolecular structures — linear terminal complexes and tetrads — are present on the plasma membrane and on membranes of the endomembrane system. The linear terminal complexes seem to be involved in the biosynthesis, assembly, and orientation of the cellulose microfibrils and the tetrads in the synthesis of the matrix polysaccharides. It is shown how the research on the supramolecular organization of cell membranes has increased the knowledge on the biosynthesis and secretion of the extracellular crystalline and non-crystalline polysaccharides in red algae. In this review, the progress to date is discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

β-Globin gene polymorphism in Saudis — triple Hpa I fragments

Summary In an attempt to investigate -globin gene polymorphism in the Saudi population, DNA samples were analysed using restriction endonucleases, Mst II and Hpa I. Both ^A and ^S genes showed extensive polymorphism and were found to be linked to 13.0 kb, 7.6 kb, 7.0 kb, and 5.6 kb Hpa I fragments. Three DNA samples out of a total of 436 analysed, had -globin gene linked to three Hpa I fragments of the sizes 13.0 kb, 7.6 kb, and 5.6 kb. In this paper we present our findings and discuss the possibility of multiple -globin loci.     

Quantitative measurement of cell-bubble attachment in bubble columns using foam flotation techniques

A simple and convenient system for quantitatively measuring the number of adsorbed animal cells per unit of bubble surface area (, unit: cells/cm2) was developed. The system was successfully applied to recombinant Chinese hamster ovary (r-CHO) suspension cultures to investigate the dynamic cell-bubble attachment in a bubble column. In serum-free medium, values increased with bubble rising height (H) and cell concentration (C) and then became constant (about 1750 cells/cm2) when H and C were sufficiently high. In medium containing protective additives, the trends of values with H were similar to that in serum-free medium. Compared with serum-free medium, polyvinyl-pyrrolidone (PVP) increased the values to 1941 cell/cm2 whereas other tested additives decreased the values of in some different degree.     

Isolation of phages infecting<Emphasis Type="Italic"> Actinoplanes</Emphasis> SN223 and characterization of two of these viruses

Phages infecting the industrially important Actinoplanes strain SN223 were isolated from soil samples collected at the shores of inland waters in Germany. The genome sizes range from 53 kb to 58 kb. Preliminary analyses revealed G+C contents comparable with the G/C bias of the host. Electron microscopy of three selected viruses displayed no obvious morphological differences, the phage heads being icosahedral and their tails non-contractible. Two of the phages (Asp2, Asp3.1) characterized in more detail are capable of provoking putative pseudolysogenic growth of the host bacterium. The carrier state for Asp2, in which cells are tightly packed with viruses, was demonstrated by electron microscopy. The latter phage is apparently widely distributed, as it was isolated from regions which are distantly located, i.e. more than 600 km apart from each other.     

Segregation analysis in hereditary retinoblastoma

Summary Segregation analysis was performed on 211 nuclear families belonging to 166 pedigrees of hereditary retinoblastoma found in a number of series which have been gathered from the literature. Bilaterally affected carriers appear homogeneous. The segregation ratio in their offspring is 0.49, and the proportion of bilateral cases among affected offspring is 0.87. Both unilaterally affected and unaffected carriers appear heterogeneous. The very low segregation ratio (0.08) in the offspring of unilateral carriers who are not detected through an affected child, suggests the possiblity of two types of carriers, high and low transmitters. The proportions of low transmitters was estimated as 0.14 among all familial unilateral carriers and as 0.45 among all detected unaffected carriers. Unilateral and unaffected high transmitters give a significantly lower segregation ratio than bilaterally affected carriers.On the one hand, the existence of these two different types of carriers provides arguments in support of the hypothesis of delayed mutation. On the other hand, the differences in penetrance among high transmitters, according to their phenotype, supports the hypothesis of host resistance. Under the two-mutation hypothesis, the possibility that the mutation rate is variable among individuals and partly genetically determined, is suggested.     

The occurrence of HT-2 toxin and other trichothecenes in Norwegian cereals

A total of 449 grain samples, 102 barley, 169 wheat and 178 oat samples were collected from different regions of Norway from 1996–1998 crops, mainly from grain loads and silos. The samples were analysed for type A and B trichothecenes, the largest groups of mycotoxins produced by the Fusarium species, by gas chromatography with mass spectrometric detection (GC-MS). Factors affecting the presence of the different trichothecenes are discussed. Deoxynivalenol (DON) and HT-2 toxin were the trichothecenes most frequently detected, followed by T-2 toxin, nivalenol, and scirpentriol, scirpentriol being detected only in seven samples (>20 g/kg).Oats were the grain species most heavily contaminated with an incidence(% >20 g/kg) and mean concentration of positive samples of 70%(115 g/kg) for HT-2 toxin, 30% (60 g/kg) for T-2 toxin, 57%(104 g/kg) for DON, and 10% (56 g/kg) for nivalenol. The corresponding values for barley were 22% (73 g/kg), 5% (85 g/kg),17% (155 g/kg) and 6% (30 g/kg), and for wheat 1.2% (20 g/kg),0.6% (20 g/kg), 14% (53 g/kg) and 0% for HT-2, T-2, DON and nivalenol, respectively. Norwegian oats were found to contain HT-2 and T-2 toxin in concentrations that might be at threat to human health for high consumers of oats. The amount of DON was significantly lower than in the crop from previous years.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genetic analysis using trans-dominant linked markers in an F2 family

Trans-dominant linked markers pairs (trans referring to the repulsion linkage phase) provide a model for inferring the F₂ progeny genotype based upon both the conditional probabilities of F₂ genotypes, given the F₂ phenotype, and prior information on marker arrangement. Prior information of marker arrangement can be readily obtained from a linkage analysis performed on marker segregation data in a family resulting by crossing the F₁ individual to a tester parent or else can be obtained directly from the gametes of the F₁, or from recombinant inbred lines. We showed that a trans-dominant linked marker (TDLM) pair can be recoded as a co-dominant megalocus when the recombination fraction, r₁, for apair of TDLMs is less than 0.05. We obtained a maximum-likelihood estimator (MLE) of the recombination frequency, r₂, between a TDLM pair and a co-dominant marker in an F₂ family using the EM algorithm. The MLE was biased. Mean bias increased as r₁ and r₂ increased, and decreased as sample size increased. The information content for r₂ was compared to the information content of dominant and co-dominant markers segregating in an F₂ family. It was almost identical with two co-dominant markers when r₁0.01 and r₂0.05. For larger values of r₁, (0.05r₁0.15) a TDLM pair provided 75%–66% of the information content of two co-dominant markers. Although dominant markers can be converted to co-dominant markers by a laborious process of cloning, sequencing, and PCR, TDLM pairs could easily substitute for co-dominant markers in order to detect quantitative trait loci (QTLs) and estimate gene action in an F₂ family.     

Chydorus ‘mutilus’ Kreis, 1921 — a postephippial form of Chydorus sphaericus (O. F. Müller, 1785)

Since 1991 faunal collections have been made from the mountain lakes of NW Slovenia. These lakes are at altitudes of between 1250 and 2150 m a. s.l., and have rich biotas, both in terms of species-richness, and faunal abundance. Amongst the animals collected were hump-backed specimens of genus Chydorus. These are identical with Chydorus mutilus, a species described from Swiss mountain lakes by Kreis (1921). The abundance of specimen in the collections, coupled with the availability of data from four successive years of sampling, allowed the detailed analysis of these populations. This also includes an examination of chitinous remains preserved in subrecent sediments.The results show that C. mutilus Kreis, 1921 actually represents a postephippial form of C. sphaericus (O. F. Müller, 1785). From the data available, it appears that the hump-backed form only occurs under certain environmental conditions. Here I discuss environmental factors having the potential to trigger the formation of hump-backed Chydorus. These findings may prove significant in palaelimnological studies, and in the reconstruction of paleotemperatures.In addition, hump-backed animals, apparently identical to European C. mutilus, have also been found in a sample taken from Lake Titicaca (Peru) in 1954. This supports the hypothesis that the hump-backed morph is an environmentally-cued ecophenotype, and not an independent taxon.     

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica

Summary. Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4,6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.Correspondence and reprints: College of Life Sciences, Peking University, Beijing 100871, Peoples Republic of China.     

Tandem mass spectrometry for characterization of unsaturated disaccharides from chondroitin sulfate,dermatan sulfate and hyaluronan

Fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from chondriotin sulfate, dermatan sulfate and hyaluronan. The positional isomers of the sulfate group of mono- and disulfated disaccharides were distinguished from each other by both positive- and negative-ion fast atom bombardment tandem mass spectra, which gave sufficient information characteristic of the isomers. The anomeric isomers of nonsulfated disaccharides were characterized by the technique in the positive-ion mode. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of trisulfated disaccharide.Abbreviations FABMS fast atom bombardment mass spectrometry - MI metastable ion - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - SIMS secondary ion mass spectrometry - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - GlcA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - UA-GalNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S-GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S-GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA2S-GalNAcDiS 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA-GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose     

DNA finger printing by oligonucleotide probes specific for simple repeats

Summary Interspersed simple repetitive DNA is a convenient genetic marker for analysis of restriction fragment length polymorphisms (RFLPs) because of the numbers and the frequencies of its alleles. Oligonucleotide probes specific for variations of the GA _C ^T A simple repeats have been designed and hybridized to a panel of human DNAs digested with various restriction enzymes. Numerous RFLPs were demonstrated in AluI and MboI digested DNA with pure GATA oligonucleotides as probes. The optimal length of the probe for RFLP analysis was 20 bases taking into account fragment lengths (1.5-7 kilobases = kb), signal to background ratio, and number of clearly evaluable RFLPs. By using different restriction enzymes individual-specific hybridization patterns (DNA fingerprints) can be established. Hypervariable simple repeat fragments are stably inherited in a Mendelian fashion. Advantages of this method are discussed.     

Oligomerizations of deoxyadenosine bis-phosphates and of their 3′-5′, 3′-3′, and 5′-5′ dimers: Effects of a pyrophosphate-linked,poly(t) analog

A pyrophosphate-linked polynucleotide analog based on thymidine 3,5 bis-phosphate (pTp) catalyzes the oligomerization of activated dimers of pdAp in the presence of MgCl₂. Although no catalysis of the oligomerization of the activated monomer (ImpdAplm) was observed in the presence of MgCl₂, there was a significant stimulation of oligomerization by the template in the presence of MnCl₂.     

Virulence as a consequence of genome instability of a novel temperate bacteriophage, ΦRsG1, of Rhodobacter sphaeroides Y

A novel temperate bacteriophage, designated RsG1, was isolated from Rhodobacter sphaeroides Y (previously designated Rhodopseudomonas sphaeroides) following exposure to mitomycin C. The phage morphology, as revealed from electron microscopy, showed a hexagonal head (90 by 46.5 nm) connected with a tail (116 by 9.4 nm), to which a collar was proximally attached. A morphologically similar phage was also produced by spontaneous lysis of the cells. While RsG1 did not grow on any other bacterial strain tested, spontaneously produced phage particles propagated (and formed plaques) on R. sphaeroides Y still carrying RsG1 in the prophage state. The genome of RsG1 consisted of double stranded linear DNA with cohesive ends and a GC-content of 71.8 mol%. The DNA molecules formed circles in vitro with a mean contour length of 17.18±0.4 m, which corresponds to a size of 49 kbase pairs (kb). On the other hand, DNA extracted from the virulent phage particles was heterogeneous and consisted of two DNA species of different size, occurring in a ratio of about 1:1. These molecules also circularized having contour lengths of 17.18±0.4 m and 14.02±0.41 m corresponding to 49 and 40 kb, respectively. Restriction digest analysis of the two DNA species and DNA from RsG1 indicated that they are similar, and allowed the indentification of an 11.5 kb EcoRI fragment that carries the cohesive ends. Because DNA from RsG1 and the 49 kb DNA of the virulent phage particles were indistinguishable with the criteria applied, it is suggested that phage particles containing the 40 kb DNA represent the virulent type of phage, termed RsG1.1.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

Different ζ globin gene deletions among Black Americans

Summary Four types of chromosomes with a deletion between the human embryonic and globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5 to the globin gene [(X⁺) or (X^-)]. The deletions are identifiable in Xba I digests of genomic DNA using an or a globin gene probe which yield fragments of 23 kb from (X⁺)–^* chromosomes or 27 kb from (X^–)–^* chromosomes. Digestion with other enzymes and probing with both and probes gave fragments typical of the two globin gene deletions previously identified in Polynesians. Among Black Americans, these globin gene deletions have been found in combination with globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult globin genes.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451