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Background: Cyclic vomiting syndrome is classified as a possible subset of migraine. Brain magnetic resonance imaging (MRI) findings of white matter hyperintensities are well documented in migraineurs, but not in patients with cyclic vomiting syndrome. This study focuses on white matter hyperintensities in children with cyclic vomiting syndrome. Methods: We investigated our database of outpatient medical records for the diagnosis codes associated with cyclic vomiting syndrome from January 2008 to October 2018. Results: Brain MRIs were obtained in 31 of 185 patients (∼17%) with a diagnosis code related to cyclic vomiting syndrome. We excluded 13 of 31 patients because of the inaccessibility of images or a confounding diagnosis. Remaining patients were divided into 2 groups: 13 of 18 cyclic vomiting syndrome with migraine (CVS+M), and 5 of 18 cyclic vomiting syndrome without migraine (CVS-M). We found that 3 of the 13 patients in the CVS +M group had migraine-like white matter hyperintensities compared to 0 of the 5 in the CVS-M group. Conclusion: This small study suggests a possible relationship between white matter hyperintensities and CVS+M. A larger study is required to validate these findings.

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

OBJECTIVE Little is known about the prevalence of attention-deficit/hyperactivity disorder (ADHD) in children with hydrocephalus. In this study, the authors assessed the prevalence of ADHD and its association with clinical and demographic factors, including intellectual disability (ID), a potential factor that can confound the diagnosis of ADHD. METHODS The authors conducted a cross-sectional study of children 6–12 years of age with hydrocephalus using parent telephone surveys. The Child and Adolescent Intellectual Disability Screening Questionnaire (CAIDS-Q) and the National Institute for Children’s Health Quality (NICHQ) Vanderbilt Assessment Scale were used to screen for ID and ADHD, respectively. Among children without ID, the authors identified those with ADHD and calculated a prevalence estimate and 95% confidence interval (Wald method). Logistic regression analysis was conducted to compare children with ADHD with those without ADHD based on demographics, family income, parental educational, etiology of hydrocephalus, and primary treatment. As a secondary analysis, the authors compared subjects with ID with those without using the same variables. Multivariable analysis was used to identify factors with independent association with ADHD and ID. RESULTS A total of 147 primary caregivers responded to the telephone questionnaire. Seventy-two children (49%) met the cutoff score for ID (CAIDS-Q). The presence of ID was significantly associated with lower family income (p < 0.001). Hydrocephalus etiology (p = 0.051) and initial treatment (p = 0.06) approached significance. Of children without ID (n = 75), 25 demonstrated a likely diagnosis of ADHD on the NICHQ, yielding a prevalence estimate of 0.33 (95% CI 0.22–0.44). No clinical or demographic variable showed significant association with ADHD. CONCLUSIONS These data indicate that the prevalence of ADHD among children with hydrocephalus (33%) is higher than among the general population (estimated prevalence in Alabama is 12.5%). ID is also common (49%). Routine screening for ADHD and ID in children with hydrocephalus may help to ensure that adequate resources are provided to optimize functional outcomes across development.

Purpose of ReviewFunctional neurological disorder (FND) is a multi-network brain disorder that encompasses a broad range of neurological symptoms. FND is common in pediatric practice. It places substantial strains on children, families, and health care systems. Treatment begins at assessment, which requires the following: themedicaltask of making the diagnosis, theinterpersonaltask of engaging the child and family so that they feel heard and respected, thecommunicationtask of communicating and explaining the diagnosis, and thelogisticaltask of organizing treatment.Recent FindingsOver the past decade, three treatment approaches—Retraining and Control Therapy (ReACT), other cognitive-behavioral therapies, and multidisciplinary rehabilitation—have been evaluated in the USA, Canada, and Australia. Of children treated in such programs, 63 − 95% showed full resolution of FND symptoms. The common thread across the programs is their biopsychosocial approach—consideration of biological, psychological, relational, and school-related factors that contribute to the child’s clinical presentation.SummaryCurrent research strongly supports a biopsychosocial approach to pediatric FND and provides a foundation for a stepped approach to treatment. Stepped care is initially tailored to the needs of the individual child (and family) based on the pattern and severity of FND presentation. The level of care and type of intervention may then be adjusted to consider the child’s response, over time, to treatment or treatment combinations. Future research is needed to confirm effective treatment targets, to inform the development of stepped care, and to improve methodologies that can assess the efficacy of stepped-care interventions.

Revealing the genetic underpinnings of cotton productivity will require understanding both the prehistoric evolution of spinnable fibers, and the results of independent domestication processes in both the Old and New Worlds. Progress toward a reference sequence for the smallest Gossypium genome is a logical stepping-stone toward revealing diversity in the remaining seven genomes (A, B, C, E, F, G, K) that permitted Gossypium species to adapt to a wide range of ecosystems in warmer arid regions of the world, and toward identifying the emergent properties that account for the superior productivity and quality of tetraploid cottons. The greatest challenge facing the cotton community is not genome sequencing per se but the conversion of sequence to knowledge.

The ketogenic diet (KD) is an established, effective nonpharmacologic treatment for intractable childhood epilepsy. The KD is provided differently throughout the world, with occasionally significant variations in its administration. There exists a need for more standardized protocols and management recommendations for clinical and research use. In December 2006, The Charlie Foundation commissioned a panel comprised of 26 pediatric epileptologists and dietitians from nine countries with particular expertise using the KD. This group was created in order to create a consensus statement regarding the clinical management of the KD. Subsequently endorsed by the Practice Committee of the Child Neurology Society, this resultant manuscript addresses issues such as patient selection, pre-KD counseling and evaluation, specific dietary therapy selection, implementation, supplementation, follow-up management, adverse event monitoring, and eventual KD discontinuation. This paper highlights recommendations based on best evidence, including areas of agreement and controversy, unanswered questions, and future research.

An intronless nuclear gene, psbT, isolated from cotton, encodes a putative 11-kDa protein (PSII-T) highly homologous in its C terminus to the N terminus of the partially sequenced PSII-T protein from spinach photosystem II. Analysis of the deduced amino acid sequence of cotton PSII-T revealed the presence of potential chloroplast stroma and thylakoid targeting domains and a putative mature PSII protein of 3.0 kDa, composed of only 28 amino acid residues. The cotton PSII-T 11-kDa precursor was synthesized in vitro in a wheat germ extract translation system, and the translation product was used in assays for protein imports into isolated pea chloroplasts. It was shown that the cotton PSII-T precursor was imported into the chloroplasts, processed to a mature form of approximately 3.0 kDa, agreeing with the predicted size from amino acid sequence analysis, and localized to the lumenal side of the thylakoid membrane, thus defining a new nuclear encoded lumenal protein and the smallest polypeptide of PSII reported to date. Processing of the PSII-T precursor occurred in two steps and involved the formation of a stromal intermediate of approximately 7.5 kDa, as predicted from primary structure analysis.

U.S. and international medical organizations recommend against testing children for genetic diseases that occur after adolescence and for which no prevention or treatment is available. Virtual Mentor is a monthly bioethics journal published by the American Medical Association.

Among the proteins that accumulate as plant seeds desiccate are several protein families that are composed principally of a tandemly repeated 11-mer amino acid motif. Proteins containing the same motif accumulate in the desiccating leaves of a desiccation-tolerant plant species. This motif is characterized by apolar residues in positions 1, 2, 5 and 9, and charged or amide residues in positions 3, 6, 7, 8 and 11. An alpha helical arrangement of the 11-mer repeating unit gives an amphiphilic helix whose hydrophobic stripe twists in a right-handed fashion around the helix. Should these proteins dimerize via binding of their hydrophobic faces, a right-handed coiled coil would be formed. Such a structure has not previously been observed. A conceivable function for these proteins in ion sequestration in the desiccated state is proposed.

Introduction Juvenile neuronal ceroid lipofuscinosis (JNCL; Batten disease) is a rare, inherited, fatal lysosomal storage childhood disorder. True for many rare diseases, there are no treatments that impact the course of JNCL. The University of Rochester Batten Center's (URBC) mission is to find treatments to slow, halt, or prevent JNCL. Objectives Our initial objective was to develop clinical research infrastructure preparatory to clinical trials, establish a JNCL research cohort, construct a disease-specific clinical outcome measure, and validate a non-invasive diagnostic sampling method. The long-term objective is to design and implement JNCL clinical trials. Methods The Unified Batten Disease Rating Scale (UBDRS) was developed. The Batten Disease Support and Research Association (BDSRA) referred participants; annual BDSRA meetings provided a mobile research setting for registry enrollment and UBDRS piloting. Neuropsychological examinations were performed, enabling external validation of the UBDRS. Buccal epithelial cell collection for genotyping was introduced. Telemedicine for remote UBDRS assessment was piloted. Results The registry enrolled 198 families representing 237 children with NCL. The UBDRS was piloted, was validated and has been used to collect natural history data from 120 subjects. Funding and regulatory approval were obtained for a recently launched phase II clinical trial. Several additional lines of inquiry were reported. Conclusion The registry and BDSRA collaboration have enabled development of a clinical rating scale, natural history and neuropsychological studies, and genetic studies for disease confirmation. This work highlights an approach for preparatory natural history research and infrastructure development needed to facilitate efficient implementation of clinical trials in rare diseases.

Over 10 years ago several families of proteins that accumulate to high levels during the maturation phase of cotton embryogenesis were identified by 2D gel electrophoresis (Dure et al. 1981). These proteins could be induced to accumulate earlier in embryogenesis when immature embryos were dissected and incubated in dilute solutions of ABA (without ABA, the embryos germinate precociously). Subsequently, cDNA representatives of many of these protein families were obtained by subtractive hybridization and identified by hybrid arrested translation (Galau et al. 1986). To reflect the developmental period of high accumulation, these protein families were named Lea, mnemonic for late embryogenesis abundant; likewise lea genes and mRNAs. By 1988 a representative gene from each of six different lea families had been sequenced and their amino acid sequence deduced (Baker et al. 1988). In the last several years a large number of gene sequences have been reported that are homologous to the sequenced lea gene families. Many were uncovered in studies designed to find genes whose expression is up-regulated in response to water stress, salt stress and cold stress. In most all cases, the high expression of these genes was found to be up-regulated by exogenous ABA as well. Furthermore, the stress-induced expression of these genes was found initially in non-seed tissues.

The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods. The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits. The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting. Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell. These values calculate to concentrations of about 226 and 283 [mu]M, respectively, in the cell aqueous phase immediately prior to seed desiccation. In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells. Thus, their function does not appear related to unique functions of specific cell or tissue types. The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.

1. Transcriptional and Post-transcriptional Regulation of Gene Expression in Plants C. Kuhlemeier. 2. Agrobacterium and Plant Genetic Engineering P.J.J. Hooykaas, R.A. Schilperoort. 3. Plant-transposable Elements and Gene Tagging A. Gierl, H. Saedler. 4. Plant and Organ Development R.F. Lyndon, D. Francis. 5. Molecular Biology of Fruit Ripening and its Manipulation with Antisense Genes J. Gray, S. Picton, J. Shabber, W. Schurch, D. Gierson. 6. Developmental Aspects of the Rhizobium-legume Symbiosis H.J. Franssen, I. Vijn, Wei Cai Yang, T. Bisseling. 7. The Molecular Biology of Disease Resistance N.T. Keen. 8. The Search for the Proteinase Inhabitor-inducing Factor, PIIF C.A. Ryan. 9. Molecular Basis of Disease Susceptibility in the Texas Cytoplasm of Maize C.S. Levings III, J.N. Siedow. 10. The Chloroplast Genome M. Sugiura. 11. The Biochemistry and Molecular Biology of Plant Lipid Biosynthesis A.R. Slabas, T. Fawcett.

The separation of isotransferring aminoacyl-tRNA synthetase activities (amino acid : tRNA ligases, EC 6.1.1.x) for several amino acids extracted from tissues of embryonic and germinating cotton seeds was carried out by DEAE-cellulose column chromatography. Evidence was obtained that the separated activities represent discrete enzymes, and could be defined as cytosol or chloroplast enzymes by several criteria. The levels of the cytosol enzymes per cell were found to be constant in germinated and ungerminated cotyledons. Chloroplast enzymes were found to be present in immature embryonic cotyledons and in roots at constant levels relative to the cytosol enzymes, but found to increase markedly in germinating cotyledons. This increase takes place to the same extent in etiolated cotyledons as in greened cotyledons indicating that the chloroplast synthetase increase is analogous to the simultaneous increase in chloroplast tRNA and rRNA which also is not light dependent. The separated cytosol and chloroplast enzymes show varying degrees of specificity for isoaccepting tRNA species from homologous and heterologous sources.

Cordycepin (3′dAdo), an inhibitor of RNA synthesis and poly(A) addition to pre-mRNA, inhibits protein synthesis and the appearance of enzyme activity thought to come from stored mRNA in germinating cotton cotyledons. 3′dCyd and Actinomycin D, other RNA synthesis inhibitors, have no effect on early germination. The period of sensitivity to 3′dAdo is short, comprising the 6th–30th hour of germination. This is consistent with the idea that the stored mRNA of cotton cotyledons is not processed until germination.

The concentrations of messenger ribonucleic acids (mRNAs) in three stages in cotton cotyledon ontogeny were examined by total poly(A)+ mRNA hybridization with unfractionated complementary DNA. This study and others show that these RNAs are representative of the total mRNA population. A novel analysis of all reciprocal hybridization reactions detected at least 17 groups of mRNAs, the sequences of which change together in concentration during this developmental period. We have defined an mRNA subset as a group or groups of mRNAs that change together in concentration in a similar fashion, regardless of the abundance of its members. About 11 such subsets are detected, several of which contain at least two groups which change in concentration in parallel. These experiments have identified the same mRNA subsets detected in a companion study of the abundant proteins synthesized in vivo and in vitro during this developmental period and indicate that similar changes occur for less abundant mRNAs as well.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome. Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ( C 0 t 1 2 pure ¶ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ( C 0 t 1 2 pure = 4.6 × 10 −4 ) and intermediately repetitive sequences constitute the remaining 27% of the genome ( C 0 t 1 2 pure = 1.46 ). Hybridization of 125I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome. The interspersion of repetitive sequences that reassociate between C0t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component. Highly repetitive sequence elements that are reassociated by a C0t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C0t = 10−4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.

The synthesis of the principal cottonseed storage proteins during embryogenesis has been followed by analyses of stained protein gels and of fluorographs of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross-reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors. Storage protein synthesis falls off markedly in late embryogenesis simultaneously with the loss of a superabundant class of mRNAs (shown by cDNA:RNA reassociation) that are presumed to be those for the storage proteins. The synthesis of these proteins ceases abruptly when immature embryos are removed from the boll and allowed to germinate precociously or when this precocious germination is prevented by incubation in abscisic acid. Thus, abscisic acid is not implicated in the expression of the storage protein genes. A scheme involving co-translational processing into vesicles, glycosylation, and slow in situ cleavage to produce the mature storage proteins is proposed.

The storage proteins of the cottonseed are composed of α and β globulins which are homologous to the vicilins and legumins, respectively, of the legumes. Gene sequences for members of both protein families have been determined, and the derived amino acid compositions are presented here. The proteolytic cleavages and glycosylations involved in the processing of the preproproteins during embryogenesis to yield the proteins found in the mature seed are outlined.

Changes in messenger ribonucleic acid (mRNA) populations during embryogenesis of cottonseed have been followed by cataloging (a) extant proteins, (b) proteins synthesized in vivo, and (c) proteins synthesized in vitro from extracted RNA, all at specific stages of embryogenesis. Evidence is presented for the existence of five mRNA subsets, all apparently under different regulatory regimes, that produce the abundant proteins of embryogenesis. One of these functions principally during the cell division phase of embryogenesis and encodes among its products the seed storage proteins whose mRNA is superabundant during this period. This subset has disappeared from the abundant group by the mature seed stage. Two other subsets appear in late embryogenesis, one of which may result from the removal of the embryo from the maternal environment, since it is inducible by excision of the young embryo from the seed. The other appears to be induced by the plant growth regulator abscisic acid, whose endogenous concentration increases at this stage. It can be induced by incubating excised young embryos in abscisic acid. The last two subsets exist throughout embryogenesis, but only one of them appears to function in germination.

DNAs complementary to the mRNAs coding for the major cotton 48 kD and 52 kD storage proteins have been cloned and used to characterize the principal cotton storage protein gene family. The principal storage proteins are found to emanate from three subsets of genes that share some homology, as shown by common antigenic determinants shared by the proteins themselves, but that are distinguishable by nucleic acid hybridization. A single sequence subfamily of 2.26 kb mRNAs codes for the 69 kD preproteins (precursors to the mature 48 kD proteins) and two sequence subfamilies of 1.96 kb mRNAs each code for 60 kD preproproteins (precursors to the mature 52 kD proteins). Hybrid arrested translation shows that cloned members of these three subfamilies hybridize only with the mRNAs of a single subfamily at moderate criterion. These three subfamilies comprise all of the principal storage protein mRNAs detectable byin vitro translation. With hybridization at low criterion, some homology has been detected between the two 1.96 kb mRNA families, although no homology has yet been detected between the 2.26 kb mRNA family and either of the two 1.96 kb mRNA families.

Any study of ribosomes and polyribosomes from the point of view of tissue development demands a quantitative extraction of all classes of these particles from the tissue. The use of bentonite, a commonly used inhibitor of RNase, has been found to influence considerably the total yield of the ribosomal complement of germinating oat tissues, and to influence the yield of monomeric ribosomes relative to polyribosomes. To prepare ribosomes and polyribosomes from these tissues, oat seeds were germinated in darkness for 72 hours after which time the coleoptile sheath and its enclosed primary leaf were excised from the plantlet at the coleoptilar node. This material, hereafter referred to as sections, was immediately homogenized with a loose fitting, motor driven Dual1 homogenizer at l-2°C. Generally 100 to ZOO sections were utilized for each preparation and homogenized in 15 to 30 ml of medium respectively. The homogenizing medium was composed of 0.1 M trissuccinate buffer, pH, 7.6 at 5"C, 0.005 M MgC12, 0.001 PI spermidine. 0.25 M sucrose and other additives as mentioned in the text. This homogenate was centrifuged for 20 minutr-, at 10,000 gravities and sodium deoxycholate (Na Dot) added to the supernatant to give a concentration of 0.5%. The supematant was next layered over a concentrated sucrose solution (5 ml over 7 ml) in Spinco #40 rotor centrifuge tubes. If the entire ribosome population Igas to be sedimented in the subsequent centrifugation, the sucrose concentration was 1 molar; if polyribosomes, predominately free of monomeric ribosomes, were to be collected, the sucrose con

Two isoaccepting chloroplastic and one cytoplasmic tRNA Met species have been separated from germinating cotton cotyledons. The methionylated form of one of the chloroplastic species (but none of the other or of the cytoplasmic tRNA Met ) can be formylated either by an endogenous transformylase or by Escherichia coli transformylase.

Cotton cotyledons have been found to synthesize de novo during germination two enzymes, a carboxypeptidase and isocitratase. The mRNA for this synthesis is transcribed in embryogenesis at a point when the embryos have reached two-thirds of their final size. The translation of the mRNA is prevented during the completion of embryogenesis by the plant hormone abscisic acid, which is produced by the tissues surrounding the embryo and absorbed by the embryo. The inhibition of the translation of the mRNA appears to require the concomitant synthesis of nonribosomal RNA. The mRNA for the two enzymes can be prematurely induced in younger cotyledons by excision of the embryo from the mother plant. This also stops DNA synthesis and cell division in this tissue. The synthesis of the mRNA and the cessation of DNA synthesis and cell division normally occur at the time in embryogenesis when the vascular connection between the mother plant and the incipient seed degenerates. It is postulated that these events are the result of a loss of vascular factors supplied by the mother plant. The synthesis of the two enzymes appears to be identically regulated, and they may represent a body of enzymes that are required for germination and whose synthesis is under the same coordinated regulation.

We have measured the amount of tRNA in cotton cotyledons that can be charged with each of the 20 amino acids at four developmental stages in cotyledon embryogenesis and germination. We have examined the number and relative levels of isoaccepting tRNA species for 15 amino acids at the same developmental stages by RPC-2 (Freon) chromatography and by the chromatography of RNAse T1 digests of aminoacyl-tRNA. We have made the same measurements with tRNA from cotton root tissue and from partially purified cotton chloroplasts. The results suggest that there are no differences in the levels of cytoplasmic tRNA species between cotyledons of various developmental stages nor between cotyledons and roots. There is, however, a large increase in the amount of chloroplastic tRNA per cell that occurs during early germination, and this increase is not induced by a light stimulus. Chloroplast tRNA exists in young embryogenic cotyledons and roots, and the amounts of the individual chloroplastic isoaccepting tRNA species do not change relative to each other as they increase in the cotyledon cells during germination. It is concluded from these data that developmental changes in cotton seedlings are not accompanied by changes in the capacity for translating individual code words.

The control of the translation inhibition of germination mRNAs during cotton embryogenesis has been studied by determining the events necessary to remove the translation inhibition during precocious germination. The results indicate that the plant hormone abscisic acid may control translation inhibition by a mechanism involving RNA synthesis.

We have sequenced cDNA clones representing each of the three distinct groups of storage proteins of the cotton seed. Characteristics of their mRNAs and derived proteins are given. Dot matrix analysis of the nucleotide and amino acid sequences shows that 2 of these groups of proteins have a great deal of vestigial homology at low stringency and should be considered subfamilies of a single storage protein gene family. The remaining group is quite distinct and should be considered a separate multigene family. It also can be divived into 2 subfamilies based on the presence or absence of glycosyl residues and other sequence differences.These proteins are processed to smaller species during embryogenesis, and all of the mature storage proteins of cotton can be traced back to these 2 gene families.In view of these relationships we propose that these 2 families be called the α and β globulins of cotton storage proteins, each comprised of an A and B subfamily.

Aspects of the embryogenesis of higher plants that are of interest to molecular and cell biologists are reviewed. What is known about the changing population of developmentally regulated mRNA is summarized, and the properties of the gene products that most distinguish embryogenesis, the nutritional storage proteins, are collated.

The composition of the free amino acid pool in embryonic cotton (Gossypium hirsutum) cotyledons is quite distinct from that of endosperm, and that of germinated, greened cotyledons is quite distinct from that of leaves. During germination (including the precocious germination of immature seeds), the pool expands considerably showing a pronounced accumulation of asparagine. The high level of asparagine found in seedling roots and in the cotyledon vascular exudate indicates that this is the major transported amino acid in germination. There is no pool expansion in the presence of abscisic acid. In the presence of actinomycin D, the pool expands, but an enormous accumulation of glutamine takes place. The composition of the pool at any stage is not related to the composition of the isoacceptor transfer RNA pool, nor to the composition of the storage protein. Anaerobiosis leads to an accumulation of aspartate, alanine, and glycine at the expense of asparagine; however, desiccation does not result in an accumulation of proline. Conspicuously high levels of arginine are maintained through embryogenesis and germination. The levels of individual amino acids are presented as nanomol per cotyledon pair and as per cent of total pool.

For the past two years we have been trying to determine the mechanisms by which the translation of the stored mRNA of cotton seeds is prevented until the commencement of germination. We have collected data that suggests that the stored mRNA is not completely processed until germination begins. More specifically, it does not appear to become polyadenylated prior to seed germination. Before presenting these data, we would like to summarize briefly the evidence for the existence of stored mRNA in cotton seeds.

Our interest in the storage proteins of the cotton seed stems from the fact that they represent the products of genes that are expressed during a specific period during seed development. Thus their expression is regulated by developmental cues, and it is the comprehension of the molecular nature of these cues that is the principle interest of our laboratory. In order to provide a background to searching for these cues, we have attempted to describe the embryogenesis of this organism in terms of changes in the population of expressed genes by following the changes in the population of mRNAs and proteins during this phase of ontogeny. We have observed other sets of gene products (mRNAs) that are expressed before, after or overlapping with the storage protein genes. Presumably each set is expressed in response to different cues that arise in the tissue at specific times in embryogenesis so as to produce the mature seed capable of successful germination. In order to use the storage protein genes as a tool in the search for the molecular bases of developmental cues, we have examined storage protein properties and biosynthesis in some detail.

Publisher Summary This chapter discusses that much of the mRNA used by germinating cotton cotyledons in the first several days of germination is transcribed in embryogenesis but not used until germination begins. It considers the body of mRNA to code for proteins unique to the germination process rather than representing mRNA functioning in embryogenesis and carried over undergraded into germination. In fact, a specific enzyme, unique to germination, arises from this “stored” mRNA. The chapter attempts to determine by several methods whether preexisting mRNA is polyadenylylated during the first 24 hours of germination. The first of these involved measuring the relative extent of incorporation of [ 32 P] phosphate and [2- 3 H] adenosine into the mRNA portion and poly (A) portion of mRNA (A,) of cotyledons during the hours 10–20 of germination. This was done by isolating mRNA (A n ) from extracts of the tissue on oligo( dT)-cellulose columns using standard techniques. The mRNA portion of the molecules was digested with ribonucleases A and T 1 and the poly (A) portion was recovered by oligo (dT)-cellulose chromatography. The chapter summarizes that the values obtained in the experiments for the true mass-average mRNA chain-length and for the relative amounts of preexisting and newly synthesized mRNA that is polyadenylylated during the first day of germination. Although there is considerable variation in the latter values depending on the method of evaluation used, in every case the polyadenylylation of preexisting RNA is suggested.

The cDNA and/or genomic DNA sequences of 13 globulin storage proteins from flowering plants (angiosperms) are now known. They represent 8 genera, 5 families and 5 orders of plants and include one monocotyledonous species. Here, the coding nucleotide and amino acid sequences of these proteins are compared by dot matrix analysis and gross protein domains visualized by hydropathy analyses. The vestigial homologies visualized by these means indicate that all of the globulin storage proteins of flowering plants have emanated from 2 genes that existed at the beginning of angiosperm evolution.A curious polypeptide domain of 150-200 amino acids located near the N terminus is found in a globulin subgroup of 2 genera widely separated phylogenetically. The domain appears to have resulted from an ancient insertion that has been deleted in most of its descendant genes.

The expression of chloroplast genes during cotton embryogenesis was followed by measuring hybridization of cloned chloroplast genomic fragments to total RNA immobilzed on membrane filters. RNA was extracted from the cotyledons of cotton embryos at different stages of development ranging from very small embryos (5 mg) to the mature dry seed and from the cotyledons of seeds germinated for several days. The clones used in the hybridization reaction were those for 4 known chloroplast genes and one chloroplast sequence whose protein product is yet to be determined; all are from organisms other than cotton. The results of these dot blot hybridizations show that these genes are expressed as RNA constitutively during embryogenesis and that this expression increased between 5 and 20-fold (depending on the genomic sequence) during germination. In some cases it appears that these genes are expressed at a higher level in very young embryos than in more mature embryos. These results are surprising since cotton cotyledons are not exposed to light during embryogenesis and are not green at any embryonic stage.The extent of artifactual hybridization signals due to the presence of ctDNA in the RNA preparations was measured by treating the RNA on the filters with DNase I prior to hybridization with a labeled probe. To determine if the increased expression of these genes observed during germination is correlated with an increase in chloroplast genomes per cell, we followed the levels of ctDNA during embryogenesis and germination by hybridization of the clones to total DNA from different developmental stages. Although ctDNA per cell increases in the cotyledons during early germination in light or dark conditions, this increase is less than the increase observed in the mRNA levels, suggesting that the transcription rate increases or the mRNA degradation rate decreases for these genes during early germination.

Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins. The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.

The enzymic basis for the flow of nitrogen from arginine to asparagine during the first 3 days of germination has been measured in extracts from cotton (Gossypium hirsutum) cotyledons. Evidence that asparagine synthetase regulates asparagine accumulation in germination (for transport to the axis) is presented. Further, evidence that the bulk of the nitrogen passed from one generation to the next in dicots is through an asparagine cycle involving the following sequence asparagine → arginine → storage protein → arginine → asparagine is discussed.

The developmental time period and the magnitude of expression of the genes for the principal cottonseed storage proteins have been measured by several means. RNA was extracted from cotton cotyledons at stages during embryogenesis and the relative amounts of the mRNAS for these proteins were determined by cell-free translation in the wheat germ system and by dot and northern hybridization of the RNA with cloned cDNA probes representing the three subfamilies of the major storage protein genes. The rates of reassociation in solution of some of the RNAs with one of the cDNA clones were also determined. Data from all four procedures show that the storage protein mRNAs are demonstrable in very small embryo cotyledons, rapidly reach a high abundance level that is maintained during most of embryo growth, and then fall precipitously in amount in the last days of embryogenesis. The expression of all three gene subfamilies appears coordinate.Further, cDNA reverse transcribed from the poly(A)(+) mRNA from a stage of maximum storage protein synthesis was hybridized to saturation with cDNA clones representing each of the subfamilies. These data indicate that the mRNAs for two of the families reach the same relative level in the total mRNA population which is about 15% of the total mRNA mass. The mRNA of the third subfamily comprises only 5% of the total mRNA mass at this stage. This apparent 3∶3∶1 ratio of mRNAs does not change during the period of storage protein synthesis. Based on the amounts of the storage protein species in the mature seed, the mRNAs of each subfamily appear to be translated to the same extent during embryogenesis.

LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plant's life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.