Parents' experiences of living with, and caring for children, adolescents and young adults with Mucopolysaccharidosis (MPS).

BACKGROUND: Many rare diseases of childhood are life-threatening and chronically debilitating, so living with a rare disease is an on-going challenge for patients and their families. MPS is one of a range of rare inherited metabolic disorders (IMDs) that come under category 3 of life-limiting conditions, where there is no curative treatment available at present. Although the study of rare diseases is increasingly novel, and of clinical importance to the population, the lack of empirical data in the field to support policy and strategy development is a compelling argument for further research to be sought. METHODS: This qualitative hermeneutic phenomenological study explored and interpreted Irish parents' experiences of living with and caring for children, adolescents and young adults with MPS and the impact of these diseases on their day to day life. A purposively selected sample of parents' attending the Irish National Centre for Inherited Metabolic Disorders was invited to participate in serial in-depth interviews. RESULTS: A total of eight parents' (n = 8) of children with a range of MPS disorders aged from 6 months to 22 years (MPS I Hurler syndrome, Scheie syndrome), MPS II (Hunter syndrome), MPS III (Sanfilipo syndrome) and MPS VI (Maroteaux-Lamy syndrome) were interviewed at three time points over a 17 month period. The main themes identified during data analysis were described as living with MPS, living with a genetic rare disease, the stigma of a rare condition, MPS as encompassing multiple diseases, Unknown future, hospital vs. home, experience of waiting, a tough road ahead, and things in their day-to-day life with MPS. They spoke of their child's Quality of Life (QoL), their healthy children's wellbeing, and for some, the impact on their own physical and psychological wellbeing. They also reflected on issues of stigmatisation and isolation in their experience of living with a child with a rare disorder. CONCLUSION: This study's findings reflect the wider literature on the impact of rare diseases, which have also indicated how caring for someone with MPS, a condition that is chronic, progressive and degenerative can impact on all dimensions of the family's life. Analysis of the findings using a hemenutic pheomenology perspecitve suggest that parents of children with MPS experience multiple cyclical movements across all five human lived existential experience, and they gradually develop ways to incorporate MPS in their day to day life. It was also evident that all the carers in this study experienced a range of uncertainties, with parents using terms such as 'no man's land' and 'future is unknown' to describe their world.

Exercise and nutrition interventions in advanced lung cancer: a systematic review.

UNLABELLED: In this systematic review, we sought to evaluate the effect of physical activity or nutrition interventions (or both) in adults with advanced non-small-cell lung cancer (nsclc). METHODS: A systematic search for relevant clinical trials was conducted in 6 electronic databases, by hand searching, and by contacting key investigators. No limits were placed on study language. Information about recruitment rates, protocol adherence, patient-reported and clinical outcome measures, and study conclusions was extracted. Methodologic quality and risk of bias in each study was assessed using validated tools. MAIN RESULTS: Six papers detailing five studies involving 203 participants met the inclusion criteria. Two of the studies were single-cohort physical activity studies (54 participants), and three were controlled nutrition studies (149 participants). All were conducted in an outpatient setting. None of the included studies combined physical activity with nutrition interventions. CONCLUSIONS: Our systematic review suggests that exercise and nutrition interventions are not harmful and may have beneficial effects on unintentional weight loss, physical strength, and functional performance in patients with advanced nsclc. However, the observed improvements must be interpreted with caution, because findings were not consistent across the included studies. Moreover, the included studies were small and at significant risk of bias. More research is required to ascertain the optimal physical activity and nutrition interventions in advanced inoperable nsclc. Specifically, the potential benefits of combining physical activity with nutrition counselling have yet to be adequately explored in this population.

Comparison of Thinopyrum intermedium derivatives carrying barley yellow dwarf virus resistance in wheat.

Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been demonstrated in wheat genetic stocks with Thinopyrum intermedium chromatin. A number of resistance-bearing translocations have been reported on chromosome arm 7DL from two independent Th. intermedium sources; one source is the addition line L1 and the other is the spontaneous substitution line P29. Another source of resistance in wheat cytogenetic stocks is available as a 2Ai(2D) substitution line. We used a set of 38 molecular markers and the available deletion stocks to compare the size of the 7DL translocations more comprehensively than has been done previously. We also compared the efficacy of BYDV resistance of the various genetic stocks both before and after transfer to a common genetic background. TC14 was confirmed as carrying the smallest translocation, replacing about 20% of the distal end of 7DL. TC5 and TC10 had 90% of the chromosome arm replaced by Th. intermedium chromatin; the proximal 10% corresponded to wheat chromatin. YW642 appeared to have the whole 7DL replaced by Th. intermedium chromatin, as confirmed by the co-dominant marker cfd68 mapping on the bin nearest the centromere. Translocation line P961341 had bins 3, 7, and 8 replaced by Th. intermedium chromatin, making this the second smallest translocation with BYDV and CYDV resistance. The translocation sizes reported here differ from some of the previous estimates. The translocated Th. intermedium segments appeared to be bigger than the replaced wheat 7DL fragments. All the resistances derived from the L1 and P29 group 7 chromosomes and the 2Ai#2 chromosome were effective in reducing the number of infected plants and the mean virus titre, regardless of the background. Some evidence is discussed suggesting the long arm of the Th. intermedium group 7 chromosome 7Ai#1 carries two resistances, the distal Bdv2 and a proximal second gene.

The management of constipation in palliative care: clinical practice recommendations.

Constipation is one of the most common problems in patients receiving palliative care and can cause extreme suffering and discomfort. The aims of this study are to raise awareness of constipation in palliative care, provide clear, practical guidance on management and encourage further research in the area. A pan-European working group of physicians and nurses with significant experience in the management of constipation in palliative care met to evaluate the published evidence and produce these clinical practice recommendations. Four potentially relevant publications were identified, highlighting a lack of clear, practical guidance on the assessment, diagnosis and management of constipation in palliative care patients. Given the limited data available, our recommendations are based on expert clinical opinion, relevant research findings from other settings and best practice from the countries represented. Palliative care patients are at a high risk of constipation, and while general principles of prevention should be followed, pharmacological treatment is often necessary. The combination of a softener and stimulant laxative is generally recommended, and the choice of laxatives should be made on an individual basis. The current evidence base is poor and further research is required on many aspects of the assessment, diagnosis and management of constipation in palliative care.

Trigenomic chromosomes by recombination of Thinopyrum intermedium and Th. ponticum translocations in wheat.

Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production world wide for which wild relatives have been the source of a number of translocations carrying resistance genes. Nevertheless, along with desirable traits, alien translocations often carry deleterious genes. We have generated recombinants in a bread wheat background between two alien translocations: TC5, ex-Thinopyrum (Th) intermedium, carrying BYDV resistance gene Bdv2; and T4m, ex-Th. ponticum, carrying rust resistance genes Lr19 and Sr25. Because both these translocations are on the wheat chromosome arm 7DL, homoeologous recombination was attempted in the double hemizygote (TC5/T4m) in a background homozygous for the ph1b mutation. The identification of recombinants was facilitated by the use of newly developed molecular markers for each of the alien genomes represented in the two translocations and by studying derived F(2), F(3) and doubled haploid populations. The occurrence of recombination was confirmed with molecular markers and bioassays on families of testcrosses between putative recombinants and bread wheat, and in F(2) populations derived from the testcrosses. As a consequence it has been possible to derive a genetic map of markers and resistance genes on these previously fixed alien linkage blocks. We have obtained fertile progeny carrying new tri-genomic recombinant chromosomes. Furthermore we have demonstrated that some of the recombinants carried resistance genes Lr19 and Bdv2 yet lacked the self-elimination trait associated with shortened T4 segments. We have also shown that the recombinant translocations are fixed and stable once removed from the influence of the ph1b. The molecular markers developed in this study will facilitate selection of individuals carrying recombinant Th. intermedium-Th. ponticum translocations (Pontin series) in breeding programs.

Replication-independent assembly of an insect virus (Tetraviridae) in plant cells.

Infectious virions of the insect RNA virus Helicoverpa armigera stunt virus (HaSV; Omegatetravirus, Tetraviridae) were assembled in cultured plant protoplasts of Nicotiana plumbaginifolia in the absence of detectable replication. Assembly of the virus, which has not been grown in cell culture, required cotransfection of a DNA plasmid expressing the HaSV capsid gene in combination with either genomic RNA or with DNA plasmids carrying the complete cDNAs to the two HaSV genomic RNAs. Each cDNA was placed under the control of the cauliflower mosaic virus 35S promoter and followed by a cis-acting ribozyme so that the resultant transcripts corresponded precisely to the two genomic RNAs. Protoplast assembly of infectious particles was confirmed by EM and bioassay of host insect larvae, which became diseased and produced virus particles confirmed as HaSV. Variant transcripts carrying nonviral sequences at either or both termini of the RNAs showed no infectivity, except for RNA2 carrying only a 3' terminal extension. No replication of HaSV in protoplasts was detected in pulse-labeling and blotting experiments. Insects showed less severe disease symptoms when fed protoplasts transfected with only the RNA1 and coat protein plasmids. The symptomatic larvae contained only RNA1 and failed to yield infectious progeny virus, suggesting that RNA1 is capable of self-replication. This novel plasmid-based system confirms that the reported sequence of HaSV represents an infective genome and establishes a procedure for the reverse genetics of a tetravirus.

Molecular characterization of a Thinopyrum intermedium group 2 chromosome (2Ai-2) conferring resistance to barley yellow dwarf virus.

The wheat--Thinopyrum intermedium addition lines Z1 and Z2 carry 21 pairs of wheat chromosomes and one pair of Th. intermedium chromosomes (2Ai-2) conferring resistance to barley yellow dwarf virus (BYDV). GISH results using the genomic DNA of Pseudoroegneria strigosa (S genome) as the probe indicated that the 2Ai-2 chromosome in Z1 and Z2 is an S-J intercalary translocation. Most of the 2Ai-2 chromosome belongs to the S genome, except for about one third in the middle region of the long arm that belongs to the J genome. The results of detailed RFLP analyses confirmed that the 2Ai-2 chromosome is extensively homoeologous to wheat group 2 chromosomes. Some new RFLP markers specific to the 2Ai-2 chromosome were identified. A RAPD marker, OP-R16(340), specific to the 2Ai-2 chromosome, was screened. We converted the RAPD marker into a sequence-characterized amplified region (SCAR) marker (designated SC-R16). The study establishes the basis for selecting translocation lines with small segments of the 2Ai-2 chromosome and localizing the BYDV resistance gene when introgressed into a wheat background.

Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin.

With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacterium tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (Assu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (Lssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. repens cv. Haifa. Transgenic T0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with Assu > CaMV35S > Lssu. Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci. During this work, we attempted to improve the efficiency of A. tumefaciens-mediated transformation of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T. repens. T-DNA transfer was in general not enhanced by sonication compared to traditional A. tumefaciens-mediated transformation. Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A. tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic clones.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barely yellow dwarf virus transmitted by Rhopalosiphum padi.

Barely yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development.

We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

Adenine methylation at dam sites increases transient gene expression in plant cells.

Escherichia coli encodes two major DNA methylation systems: dam, which produces 6-methyladenine; and dcm, which produces 5-methylcytosine. About 1-2% of adenine and cytosine residues in plasmid DNAs prepared in E. coli are methylated by these systems. Since DNA methylation profoundly influences gene expression in eukaryotes, we were interested in determining whether these bacterially encoded modifications might also effect plant gene expression in experimental systems. We therefore examined the influence of dam and dcm methylation on gene expression from four GUS fusion constructs in transient assays in protoplasts and microprojectile-bombarded whole tissues. In these constructs, GUS expression was driven by promoter regions derived from the Arabidopsis alcohol dehydrogenase (Adh1), maize ubiquitin (Ubi1), rice actin (Act1) and CaMV 35S genes. We show that methyladenine produced by dam methylation increased gene expression from constructs based on the Adh1, Ubi1 and Act1 genes. The increase in gene expression ranged from three-fold for Ubi1 and Adh1 in protoplasts to 50-fold for Act1 in bombarded wheat tissues. Expression of a 35S.GUS construct was, however, insensitive to dam methylation. dcm methylation had little if any effect on transient gene expression for any of these constructs. We provide indirect evidence that the critical sites of adenine methylation lie within sequences from the promoter regions, suggesting that dam methylation increases transcription rate. These results have important experimental implications and also raise the intriguing possibility that methyladenine might play a role in the regulation of gene expression in vivo.

Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances.

Zhong 5 is a partial amphiploid (2n = 56) between Triticum aestivum (2n = 42) and Thinopyrum intermedium (2n = 42) carrying all the chromosomes of wheat and seven pairs of chromosomes from Th. intermedium. Following further backcrossing to wheat, six independent stable 2n = 44 lines were obtained representing 4 disomic chromosome addition lines. One chromosome confers barley yellow dwarf virus (BYDV) resistance, whereas two other chromosomes carry leaf and stem rust resistance; one of the latter also confers stripe rust resistance. Using RFLP and isozyme markers we have shown that the extra chromosome in the Zhong 5-derived BYDV resistant disomic addition lines (Z1, Z2, or Z6) belongs to the homoeologous group 2. It therefore carries a different locus to the BYDV resistant group 7 addition, L1, described previously. The leaf, stem, and stripe rust resistant line (Z4) carries an added group 7 chromosome. The line Z3 has neither BYDV nor rust resistance, is not a group 2 or group 7 addition, and is probably a group 1 addition. The line Z5 is leaf and stem rust resistant, is not stripe rust resistant, and its homoeology remains unknown.

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.

Proanthocyanidins inhibit hydrolysis of leaf proteins by rumen microflora in vitro.

Proanthocyanidins (condensed tannins; PA) purified from the leaves of forage legumes Trifolium arvense, Lotus pedunculatus, Lotus corniculatus, Dorycnium rectum, Coronilla varia, Onobrychis viciifolia, or Hedysarum coronarium, were added to soluble lucerne (Medicago sativa) leaf protein and incubated with strained rumen fluid in vitro. Fractions were collected and frozen immediately. Denatured proteins were fractionated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), stained, and relative levels were quantified by densitometry. In the absence of PA the large subunit (LSU) of ribulose bisphosphate carboxylase (EC 4.1.1.39) was susceptible to proteolysis by rumen microflora but the small subunit (SSU) resisted breakdown. PA purified from Onobrychis was added to soluble leaf protein, at PA: protein ratios between 1:1 and 1:20. The rate of proteolysis of LSU was significantly reduced at PA: protein ratios of 1:2 and 1:1 (P < 0.001) and the rate of digestion was reduced by between 3- and 21-fold. In separate experiments PA isolated from the range of species described was added to rumen fluid to give PA: protein ratios of 1:5. The addition of PA significantly reduced the rate of proteolysis of LSU, when compared with PA-free control. There were only small differences between PA from different species. The inhibitory effect of PA may have been due to PA binding to the dietary protein or to the rumen proteases, interfering with the action of proteases on susceptible sites within the substrate.

Varying chromosome composition of 56-chromosome wheat x Thinopyrum intermedium partial amphiploids.

Thinopyrum intermedium (2n = 42) is a source of many potentially useful genes for wheat improvement. Many partial amphiploids have been produced between Th. intermedium and Triticum aestivum that are fertile and stable. These partial amphiploids all have 56 chromosomes, including seven pairs of chromosomes from Th. intermedium. To explore the genomic composition of these lines, meiotic analysis was conducted on 32 hybrid combinations between eight different partial amphiploids. All but two of the chosen parents were distinguishable on the basis of perenniality, head morphology, and reactions to leaf, stripe, and stem rusts and to barley yellow dwarf virus. Chromosome pairing in the hybrids clearly indicated that all but two of the partial amphiploids differed in their composition of Thinopyrum chromosomes. The differences varied from one to five chromosomes. This confirms molecular evidence that the extra genome of the octoploid partial amphiploids is a variable synthetic genome combining chromosomes of the three Thinopyrum genomes E, J, and X. Though the extra synthetic genomes vary widely between different octoploids, they are nevertheless stable once formed. It is argued that the failure to establish these octoploid amphiploids as a new crop is a consequence of their differing chromosome complements, which makes it impractical to interbreed them.

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa, lucerne) and Onobrychis viciifolia Scop. (sainfoin).

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the "recipient" parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33-78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.

Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues.

The UV resonance Raman spectra of horse and sperm whale myoglobin excited at 240 nm show bands between 600 and 1700 cm-1 which derive from tyrosyl and tryptophyl residues. No significant contribution from phenylalanine and peptide backbone vibrations occurs at this excitation wavelength. We examine the pH dependence of the UV resonance Raman and UV absorption difference spectra of these myoglobins to correlate the local protein environment of the tyrosyl residues as given by the protein crystal structure to their pKa values, molar absorptivities, and Raman cross sections. Some of our pKa values for the tyrosinate residues of horse Mb differ from those of previous studies. We show that the lambda max values, the molar absorptivities, and the Raman cross sections are sensitive to the local environment of the tyrosinate residues in the protein. We relate differences in the tyrosyl absorption spectra to differences in Raman cross sections. In addition, we discuss the importance to the Raman cross sections of the local electromagnetic field enhancement due to the dielectric environment of the tyrosinate residues in the protein. This local field should scale the Raman cross sections in a way useful as a probe of the average aromatic amino acid residue environment.

Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus.

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I-mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.