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PubMed2004

A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes.

BACKGROUND: Sequencing the genomes of multiple, taxonomically diverse eukaryotes enables in-depth comparative-genomic analysis which is expected to help in reconstructing ancestral eukaryotic genomes and major events in eukaryotic evolution and in making functional predictions for currently uncharacterized conserved genes. RESULTS: We examined functional and evolutionary patterns in the recently constructed set of 5,873 clusters of predicted orthologs (eukaryotic orthologous groups or KOGs) from seven eukaryotic genomes: Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Encephalitozoon cuniculi. Conservation of KOGs through the phyletic range of eukaryotes strongly correlates with their functions and with the effect of gene knockout on the organism's viability. The approximately 40% of KOGs that are represented in six or seven species are enriched in proteins responsible for housekeeping functions, particularly translation and RNA processing. These conserved KOGs are often essential for survival and might approximate the minimal set of essential eukaryotic genes. The 131 single-member, pan-eukaryotic KOGs we identified were examined in detail. For around 20 that remained uncharacterized, functions were predicted by in-depth sequence analysis and examination of genomic context. Nearly all these proteins are subunits of known or predicted multiprotein complexes, in agreement with the balance hypothesis of evolution of gene copy number. Other KOGs show a variety of phyletic patterns, which points to major contributions of lineage-specific gene loss and the 'invention' of genes new to eukaryotic evolution. Examination of the sets of KOGs lost in individual lineages reveals co-elimination of functionally connected genes. Parsimonious scenarios of eukaryotic genome evolution and gene sets for ancestral eukaryotic forms were reconstructed. The gene set of the last common ancestor of the crown group consists of 3,413 KOGs and largely includes proteins involved in genome replication and expression, and central metabolism. Only 44% of the KOGs, mostly from the reconstructed gene set of the last common ancestor of the crown group, have detectable homologs in prokaryotes; the remainder apparently evolved via duplication with divergence and invention of new genes. CONCLUSIONS: The KOG analysis reveals a conserved core of largely essential eukaryotic genes as well as major diversification and innovation associated with evolution of eukaryotic genomes. The results provide quantitative support for major trends of eukaryotic evolution noticed previously at the qualitative level and a basis for detailed reconstruction of evolution of eukaryotic genomes and biology of ancestral forms.

Animals↗

PubMed2005

Widespread correlations between dominance and homozygous effects of mutations: implications for theories of dominance.

The dominance of deleterious mutations has important consequences for phenomena such as inbreeding depression, the evolution of diploidy, and levels of natural genetic variation. Kacser and Burns' metabolic theory provides a paradigmatic explanation for why most large-effect mutations are recessive. According to the metabolic theory, the recessivity of large-effect mutations is a consequence of a diminishing-returns relationship between flux through a metabolic pathway and enzymatic activity at any step in the pathway, which in turn is an inevitable consequence of long metabolic pathways. A major line of support for this theory was the demonstration of a negative correlation between homozygous effects and dominance of mutations in Drosophila, consistent with a central prediction of the metabolic theory. Using data on gene deletions in yeast, we show that a negative correlation between homozygous effects and dominance of mutations exists for all major categories of genes analyzed, not just those encoding enzymes. The relationship between dominance and homozygous effects is similar for duplicated and single-copy genes and for genes whose products are members of protein complexes and those that are not. A complete explanation of dominance therefore requires either a generalization of Kacser and Burns' theory to nonenzyme genes or a new theory.

Gene Deletion↗

PubMedDate not supplied

[Mutations in the methylene-tetrahydrofolate reductase gene and Down syndrome].

Down syndrome (DS) is a complex genetic and metabolic disorder attributed to the presence of three copies of chromosome 21. The extra chromosome derives from the mother in 93% of cases and is due to abnormal chromosome segregation during meiosis (nondisjunction). Except for advanced age at conception, maternal risk factors for meiotic nondisjunction are not well established. A recent preliminary study suggested that abnormal folate metabolism and the 677 (C-->T) mutation in the methylene-tetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for DS. Frequency of the MTHFR 677 (C-->T) and 1298 (A-->C) mutations was evaluated in 36 mothers of children with DS and in 200 controls. The results are consistent with the observation that the MTHFR 677 (C-->T) and 1298 (A-->C) mutations are more prevalent among mothers of children with DS than controls. In addition, the most prevalent genotype was the combination of both mutations. The results suggest that mutations in the MTHFR gene are associated with maternal risk for DS

Adolescent↗

PubMed2005

Hypoxia-induced BAX overexpression and radiation killing of hypoxic glioblastoma cells.

One major challenge in treating glioblastoma multiforme (GBM) has been the presence of radiation-resistant hypoxic cells. The pro-apoptosis protein BAX has been reported to be a possible radiation sensitizer of cancer cells; however, to our knowledge, no studies have reported on the effects of BAX on radiation sensitivity under hypoxic conditions. Therefore, in this study, we specifically overexpressed murine Bax in hypoxic cells in an attempt to enhance radiation-induced cell killing. We have previously stably transfected U-251 MG and U-87 MG human GBM cells with constructs containing murine Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible factor 1 (HIF1) forms and binds to HRE; this binding facilitates the transcription of downstream genes. In the experiments reported here, two protocols were used. In the first protocol, parent and clone cells were exposed to graded doses of X rays under hypoxic conditions, left hypoxic for 0, 4, 16 or 24 h, and then assayed for clonogenic cell survival. In the second protocol, cells were incubated under hypoxic conditions for 20 h, irradiated with graded doses under hypoxia, then left in hypoxic conditions for 4 h before being assayed for clonogenic cell survival. Western blots showed that we had successfully increased Bax expression in both U-251 MG and U-87 MG Bax clone cells after 16 h of hypoxic incubation, yet dose-response curves showed no difference in radiation-induced cell killing between control non-Bax-expressing pNeo clone cells and the U-251 MG Bax clone cells using either protocol. In U-87 MG cells, the first protocol showed no difference in radiation response between control pNeo clone cells and the Bax clone, similar to the results obtained in U-251 cells. However, the second protocol revealed that Bax overexpression did render these cells more sensitive to radiation under hypoxic conditions. Therefore, we conclude that whether Bax is a radiation enhancer under hypoxia not only is cell line-dependent but also depends on when the Bax overexpression occurs.

Adaptation, Physiological↗

PubMedDate not supplied

Protein glycosylation in rat fibroblast cells expressing deletion variants of the human hsp70 gene.

Elevated heat resistance is often associated with high cellular levels of hsp70. In the rat cell line, M21, increased heat resistance is associated with both the expression of an intact exogenous human hsp70 gene, and of an exogenous stress glycoprotein, GP62. In this study, we examined heat-stress induced alterations in protein glycosylation in two variant lines of M21 that contain specific deletions in the exogenous human hsp70 gene. The deletion mutant, MVH delta Sm, lacks the nucleolar localization sequence for human hsp70 gene, whereas the other mutant, MVH delta Bg, is characterized by deletion of the ATP binding domain in the human hsp70 gene. After heat induction, the MVH delta Sm mutants exhibited constitutive and heat-induced endogenous hsp70 mRNA levels, protein glycosylation, as well as hsp70 and GP62 protein levels comparable to those in the parent cell line, Rat-1. Cellular heat sensitivity of MVH delta Sm mutants was also similar to that of Rat-1 cells, although the mutant cells had a reduced capacity for thermotolerance development. On the other hand, MVH delta Bg mutants before thermotolerance induction, exhibited constitutive endogenous and human exogenous hsp70 mRNA levels similar to those in MVH delta Sm mutants. However, after thermotolerance induction, MVH delta Bg mutants showed lower levels of heat-induced endogenous hsp70 mRNA than MVH delta Sm mutants, an overall reduction in protein glycosylation, low hsp70 and GP62 levels, and increased heat sensitivity when compared to the parent Rat-1 cell line. Incorporation of D-[2-3H]mannose into oligosaccharide precursor pools and glycoproteins was consistent with protein glycosylation pattern for each cell line. Acute heat stress resulted in the selective glycosylation of the 'prompt' glycoprotein, P-SG64, in the two deletion mutants, similar to that in M21 cells expressing the intact human hsp70 gene. The data indicate that both protein glycosylation and hsp70 expression generally correlate with cellular heat resistance and thermotolerance expression. The presence of full or partially deleted copies of human hsp70 modulates thermotolerance and protein glycosylation in a complex manner that is not yet fully understood.

Animals↗

PubMed2004

[Use of IS6110 polymorphism analysis in molecular epidemiological studies of tuberculosis (Belgrade study results)].

Tuberculosis (TB) remains a major health problem worldwide. One of the main approaches to tackling TB today is the control of transmission of the disease through monitoring the transmission of specific strains of Mycobacterium tuberculosis. In the past, efforts to type strains of M. tuberculosis were hampered by the lack of strain-specific phenotypic markers. In recent years, novel approaches to studying the epidemiology of TB have been provided by molecular biological techniques based on DNA fingerprinting. The most widely used polymorphic marker is the transposable element IS6110, which is an insertion sequence found throughout M. tuberculosis complex. It is a highly polymorphic marker which varies in both copy number and location in the M. tuberculosis genome. A standardized methodology for IS6110 DNA fingerpriniting of M. tuberculosis exploits restriction fragment length polymorphism analysis. To date, IS6110 fingerprinting has been successfully used to trace the TB transmission in different regions and populations, to quantify the relative contribution of recent infection to the TB population burden, to answer the question of endogenous reactivation versus exogenous reinfection, to trace small-scale outbreaks of TB, to analyze spread of drug-resistant and multidrug resistant strains, to confirm laboratory cross-contaminations, etc. The objective of this study was to illustrate the basic principles of molecular epidemilological studies of TB. Results of the study, which provided the first insight into the status of TB in Belgrade based on implementation of molecular methods, were used as an example.

DNA Fingerprinting↗

PubMed1984

The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis.

When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.

Animals↗

PubMedDate not supplied

[The stress gene in hypertension].

Thermosensitivity has been demonstrated in hypertensive rats and mice, and hypertension is frequent in hyperthermia-susceptible pigs. We have demonstrated that thermosensitivity segregates with hypertension in mice as a recessive trait in a single locus termed Tms. Since thermosensitivity can be demonstrated in cells obtained from neonatal hypertensive animals and persists after several passages in culture, it was of interest to study its cellular determinants. We undertook studies of candidate genes of cellular thermosensitivity, hsp70 being the major heat stress gene. First, we have observed an enhanced hsp70 mRNA accumulation in the hypertensive rat, mouse and human after heat shock of the whole animal, of its isolated organs or cultured cells. This increased accumulation of hsp70 mRNA in hypertension is due to its increased transcriptional rate. A restriction fragment length polymorphism (RFLP) of hsp70 was documented in hypertensive rats, which allowed the study of the segregation of this locus with hypertension in recombinant inbred strains of rats. At least one copy of hsp70 has been localized in RT1, the major histocompatibility complex of the rat, with polymorphism demonstrated with BamHI outside of the coding region of hsp70. This polymorphism segregates with 15 mmHg of systolic blood pressure. More recently, our studies led us to identify a polymorphism in another heat stress gene, hsp27. This polymorphism has been identified by PCR SSCP and is located in the 3' region, close to the termination translation codon.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Complexity of nuclear and polysomal RNA from squid optic lobe and gill.

The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 X 10(8) and 8.8 X 10(7) nucleotides, respectively. Approximately 80-85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single-copy DNA, values that were 2.5-fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Animals↗

PubMed2000

Many unbalanced translocations show duplication of a translocation participant. Clinical and cytogenetic implications in myeloid hematologic malignancies.

If a translocation is followed by loss of one of the two derivative chromosomes, the result is an unbalanced translocation, showing monosomy for the segments making up the lost derivative. We have found that in most unbalanced translocations, a third event takes place: a morphologically normal copy of one of the two translocation participants is added to the karyotype. This creates a complex abnormal karyotype with monosomy, disomy, and trisomy for different segments of the translocation participants. We have examined 82 unbalanced translocations from 77 patients, 73 of whom had a myeloid hemopoietic malignancy. Acquisition of a normal copy of a translocation participant was found in 49 translocations. Twenty-five of these showed trisomy for 1q. In 16 of the 25 1q-trisomic cases the translocation was t(1;7)(q10;p10) (trisomy for 1q and monosomy for 7q). Patients with trisomy for 1q were younger than the remaining patients. Whereas those with t(1;7))(q10;p10) showed brief survivals, those with trisomy 1q but monosomy for regions other than 7q survived longer than the remaining patients. We conclude that most unbalanced translocations involve a partial trisomy, that 1q is trisomic far more frequently than any other segment, and that partial trisomy is associated with patient age and survival.

Acute Disease↗

PubMed2001

Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation.

Carney complex is a multiple neoplasia syndrome featuring cardiac, endocrine, cutaneous, and neural tumors, as well as a variety of pigmented lesions of the skin and mucosae. Carney complex is inherited as an autosomal dominant trait and may simultaneously involve multiple endocrine glands, as in the classic multiple endocrine neoplasia syndromes 1 and 2. Carney complex also has some similarities to McCuneAlbright syndrome, a sporadic condition that is also characterized by multiple endocrine and nonendocrine tumors. Carney complex shares skin abnormalities and some nonendocrine tumors with the lentiginoses and certain of the hamartomatoses, particularly Peutz-Jeghers syndrome, with which it shares mucosal lentiginosis and an unusual gonadal tumor, large-cell calcifying Sertoli cell tumor. Careful clinical analysis has enabled positional cloning efforts to identify two chromosomal loci harboring potential candidate genes for Carney complex. Most recently, at the 17q22-24 locus, the tumor suppressor gene PRKAR1A, coding for the type 1alpha regulatory subunit of PKA, was found to be mutated in approximately half of the known Carney complex kindreds. PRKAR1A acts a classic tumor suppressor gene as demonstrated by loss of heterozygosity at the 17q22-24 locus in tumors associated with the complex. The second locus, at chromosome 2p16, to which most (but not all) of the remaining kindreds map, is also involved in the molecular pathogenesis of Carney complex tumors, as demonstrated by multiple genetic changes at this locus, including loss of heterozygosity and copy number gain. Despite the known genetic heterogeneity in the disease, clinical analysis has not detected any corresponding phenotypic differences between patients with PRKAR1A mutations and those without. This article summarizes the clinical manifestations of Carney complex from a worldwide collection of affected patients and also presents revised diagnostic criteria for Carney complex. In light of the recent identification of mutations in the PRKAR1A gene, an estimate of penetrance and recommendations for genetic screening are provided.

Adrenal Cortex Neoplasms↗

PubMed2000

An associational model of birdsong sensorimotor learning I. Efference copy and the learning of song syllables.

Birdsong learning provides an ideal model system for studying temporally complex motor behavior. Guided by the well-characterized functional anatomy of the song system, we have constructed a computational model of the sensorimotor phase of song learning. Our model uses simple Hebbian and reinforcement learning rules and demonstrates the plausibility of a detailed set of hypotheses concerning sensory-motor interactions during song learning. The model focuses on the motor nuclei HVc and robust nucleus of the archistriatum (RA) of zebra finches and incorporates the long-standing hypothesis that a series of song nuclei, the Anterior Forebrain Pathway (AFP), plays an important role in comparing the bird's own vocalizations with a previously memorized song, or "template." This "AFP comparison hypothesis" is challenged by the significant delay that would be experienced by presumptive auditory feedback signals processed in the AFP. We propose that the AFP does not directly evaluate auditory feedback, but instead, receives an internally generated prediction of the feedback signal corresponding to each vocal gesture, or song "syllable." This prediction, or "efference copy," is learned in HVc by associating premotor activity in RA-projecting HVc neurons with the resulting auditory feedback registered within AFP-projecting HVc neurons. We also demonstrate how negative feedback "adaptation" can be used to separate sensory and motor signals within HVc. The model predicts that motor signals recorded in the AFP during singing carry sensory information and that the primary role for auditory feedback during song learning is to maintain an accurate efference copy. The simplicity of the model suggests that associational efference copy learning may be a common strategy for overcoming feedback delay during sensorimotor learning.

Algorithms↗

PubMed2001

A histone fold TAF octamer within the yeast TFIID transcriptional coactivator.

Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator. Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components. Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo. Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin.

Alleles↗

PubMed1990

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L.

The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3' flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz' alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz' alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz' alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz' revertant, Bz':107, arose from excision of a more complex 13 kb Ds element.

Alleles↗

PubMed1976

Structural gene sets active in embryos and adult tissues of the sea urchin.

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy 3H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy 3H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 X 10(6) nucleotides. In units of 10(6) nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA: 2.1 and 3.7; coelomocyte mRNA: 3.5 and less than or equal to 1.4; tubefoot mRNA: 2.7 and less than or equal to 0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and less than or equal to 0.6; exogastrula mRNA: 14 and less than or equal to 0.6. The total complexity of each mRNA polulation is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 X 10(6) nucleotides, appears to be shared by several of the tissues studied.

Animals↗

PubMed2005

The AP-3 clathrin-associated complex is essential for embryonic and larval development in Caenorhabditis elegans.

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.

Adaptor Protein Complex 3↗

PubMed1992

Identification of subunits required for the catalytic activity of the F1-ATPase.

F1 (alpha beta) complexes containing equimolar ratios of the alpha and beta subunits have been shown to function as active ATPases, whereas individually isolated alpha and beta subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F1-ATPase activity. The minimal F1 (alpha beta)-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F1 or alpha 3 beta 3 gamma complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F1-ATPase the presence of the F1-gamma subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.

Bacteria↗

PubMed1989

The Cucurbita maxima ribosomal DNA intergenic spacer has a complex structure.

The nucleotide (nt) sequence of the 5508-nt intergenic spacer (IGS), between the 25S- and the 18S-coding regions of Cucurbita maxima rDNA, was determined. The fragment sequenced is 6142 nt long and includes 472 nt of 25S- and 162 nt of 18S-coding regions. The IGS has a complex primary structure, composed of five repetitive families (A-E) and three unique domains. It is dominated by the presence of nine, tandemly-repeating units of approximately 250 nt (repeat D), each unit containing four copies of an internal subrepeat (repeat E). The repetitive units show sequence variability consisting of nt changes, insertions and deletions. Upstream of the nine D repeats and between two copies of the B repeat is a 575-nt region, highly G + C rich (83%) and heavily biased toward C (58%) in the sense strand. Within this region are six repetitive units, averaging 42 nt (repeat C) each, containing but a single A nt. Downstream from the terminus of the 25S-coding sequence, are two tandem copies of the 103-nt A repeat. The IGS of C. maxima is longer and more complex than that of other plant IGSs described to date. The 600 nt at the 5' portion of cucurbit IGS is more conserved in evolution than the remainder, as revealed by comparison of C. maxima and C. pepo IGS restriction maps and by nucleotide sequence comparison of C. maxima and Cucumis sativa IGSs.

Base Sequence↗