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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.

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.

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 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.

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.

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.

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.

The two principal storage proteins of the cottonseed have been purified and partially characterized. They have apparent molecular weights of 52,000 and 48,000 and are both comprised of sets of isoelectric isomers. Both sets have nearly identical solubility properties, ultraviolet absorption spectra, and contain similar proportions of amino acid residues. Furthermore, both sets share antigenic determinants suggesting their origin from a common ancestral gene. However, only the 52,000 set appears to be glycosylated.

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.