Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence.

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.

Beta-sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate.

The sarcoglycan complex is involved in the etiology of four autosomal recessive limb-girdle muscular dystrophies (LGMD2C-F). A missense mutation (T151R) in the beta-sarcoglycan gene on chromosome 4q12 has been shown to cause a mild form of LGMD2E in 11 families from a Southern Indiana Amish community sharing a common haplotype. We now report that two sibs from another Amish family with mild LGMD2E are compound heterozygotes for chromosome 4q12 markers. In order to characterize the genetic defect in this new family, we determined the genomic organization of the beta-sarcoglycan gene. A second missense mutation (R91C) has now been identified in this LGMD2E Amish family. This mutation is also present in the homozygous state in another family of probable Amish ancestry. Finally, analysis of all the components of the dystrophin-glycoprotein complex was performed for the first time on a biopsy from a patient homozygous for the beta-sarcoglycan mutation (T151R). Interestingly, in addition to the loss of the entire sarcoglycan complex, we detected a reduction of alpha-dystroglycan which suggests a role for the sarcoglycan complex in stabilizing alpha-dystroglycan at the sarcolemma.

Identification of muscle-specific calpain and beta-sarcoglycan genes in progressive autosomal recessive muscular dystrophies.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants.

The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene. The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene.

Cytoplasmic DNA variation and relationships in cereal genomes.

Chloroplast (cp) and mitochondrial (mt) DNAs were isolated from four cereal genomes (cultivated wheat, rye, barley and oats) and compared by restriction nuclease analysis. Cleavage of cp and mt DNAs by Sal I, Kpn I, Xho I and EcoR I enzymes indicated that each cereal group contains specific cytoplasmic DNAs. A phylogenetic tree of cereal evolution has been obtained on the basis of cp DNA homologies. It is suggested that wheat and rye diverged after their common ancestor had diverged from the ancestor of barley. This was preceded by the divergence of the common ancestor of wheat, rye and barley and the ancestor of oats.The molecular weight of the different cp DNAs was determined from the Sal I and Kpn I patterns. cp DNAs from wheat, rye, barley and oats appeared to be characterized by a very similar molecular weight of about 80-82.10(6) d.In the case of the mt DNAs, the great number of restriction fragments obtained with the restriction enzymes used prevented precise comparisons and determination of molecular weights.

Comparison of the organization and expression of mtDNA of fertile and male-sterile sugar beet varieties (Beta vulgaris L.).

Analysis of minicircle occurrence in different samples of sugar beet mitochondrial (mt) DNA invalidates the postulated relationship between cytoplasmic male sterility (CMS) phenotype and the absence of minicircle c and d. In high molecular weight mt DNA, two types of restriction patterns are found for fertile genomes and only one type for the CMS; in spite of the multiplicity of crosses carried out by plant breeders, all the CMS varieties analyzed seem to have derived from the original cytoplasm discovered by Owen in 1945. Southern hybridizations with mitochondrial genes coding for cytochrome oxidase subunits II and III, ATPase subunits α, 6 and 9 and 26S ribosomal RNA indicate that gene organization is different between fertile and sterile genomes but similar in all fertile genomes. Transcription analysis with the same genes indicate several differences between fertile and sterile varieties but also within some fertile varieties. These results suggest that the mt genome found in male-sterile sugar beet may originate not from modifications of the fertile mitochondrial genome but from a particular source of cytoplasm, of which a possible origin is discussed.

Evidence for homologous recombination between repeated sequences containing 18S and 5S ribosomal RNA genes in wheat mitochondrial DNA.

Closely linked genes for 18S and 5S rRNAs have been located on four different cloned SalI restriction fragments of wheat (Triticum aestivum L.) mitochondrial DNA. Restriction analysis has revealed that in each of the cloned fragments, the 18S and 5S rRNA genes are contained within the same basic structural unit, R, which is at least 4 kbp long. This unit is flanked by sequences designated u (0.8 kbp), v (13.7 kbp), w (0.7 kbp), and y (1.4 kbp), in the orientations v-R-w, v-R-y, u-R-w, and u-R-y in the four different SalI fragments. We conclude that 18S + 5S rRNA genes are located at several distinct sites in the wheat mitochondrial genome, and suggest that reciprocal intra- and/or intermolecular recombination between such repeated sequences could promote extensive genomic rearrangement and thus contribute to the physical heterogeneity that is a hallmark of most plant mitochondrial DNAs.

ATP synthase subunit c/III/9 gene sequences as a tool for interkingdom and metaphytes molecular phylogenies.

The 38 sequences of the ATPase c/III/9 gene determined in bacteria, fungi, mammals, and higher plants have been used to construct phylogenetic trees by distance matrix and parsimony methods (checked by bootstrapping); alignments have been performed on the deduced amino-acid sequences and then transferred back to the nucleotide sequences. Three lineages stand out: (1) eubacteria (except cyanobacteria and alpha purple bacteria), (2) chloroplasts, together with cyanobacteria, and (3) mitochondria together with nuclei and alpha purple bacteria. The clear monophyly of the mitochondrial/nuclear lineage, taken all together, strongly suggests that the nuclear copies of the gene now residing in the eukaryotic nucleus originate from a mitochondrial transfer. Within this lineage, metaphytes emerge late and as a cohesive group, after fungi (as a dispersed group) and metazoa, yielding an order that markedly differs from that obtained through typical RNA nuclear molecules. The possible biphyletic origin of mitochondria based on mitochondrial rRNA sequences is not evidenced by these sequences. Internal branches within both the chloroplastic and the mitochondrial lineages are consistent with botanical evolutionary schemes based on morphological characters. In spite of its relatively small size, the ATPase c/III/9 gene therefore displays remarkable properties as a phylogenetic index and adds a new tool for molecular evolutionary reconstructions, especially within the metaphytes.

Characterisation of mycobacteriophage ATCC 11759. Unusual physiocochemical properties of its DNA.

Growth conditions and a purification procedure for mycobacteriophage ATCC 11759, a lytic phage for Mycobacterium smegmatis, are described. The phage is a large DNA phage with a very long tail (240 nm). A study of its DNA revealed three interesting features. 1. After denaturation the DNA molecule yields two strands of different buoyant densities. 2. The native DNA has unusual physical properties: its buoyant density in CsC1 is very low (1.654 g/cm3), its sedimentation rate is lower than expected for the molecular weight, its thermal stability at low ionic strength is high. 3. The DNA (in its native form or after reannealing) is resistant to various restriction endonucleases.

Characterization of two minicircular plasmid-like DNAs isolated from date-palm mitochondria.

We report here the identification and characterization of two minicircular plasmid-like DNAs isolated from mitochondria of a moroccan date-palm variety. Both molecules were cloned and used as probes in Southern analyses of mitochondrial and total-cellular DNA. Evidence was obtained that these plasmid-like DNAs cross-hybridized but did not show any homology to nuclear, chloroplastic, or main mitochondrial genomes. Sequence analysis revealed that both minicircles, 1,346- and 1,160-bp long, share several stretches of homology, the most important consisting of three identical clusters of lengths 42, 47 and 38 bp. In contrast, no major homology was observed with the other higher-plant plasmid-like DNAs reported so far. Sequence analysis also revealed the presence, in the same strand of one of the minicircles, of two open reading frames potentially encoding proteins 89 and 86 amino acids in length. Interestingly, Northern analyses, using single strands of each minicircle as probes, showed the presence of two transcripts hybridizing only with the strand bearing these two open reading frames. However, computer-assisted comparison of the predicted polypeptide sequences with a protein-sequence library failed to detect any significant homology to known sequences.

Gametoclonal variation detected in the nuclear ribosomal DNA from doubled haploid lines of a spring wheat (Triticum aestivum L., cv. 'César').

The organization of the nuclear ribosomal DNA from a parental line of wheat (Triticum aestivum L., cv. 'César') and its anther-derived first cycle and second cycle doubled haploid lines has been analyzed by DNA-DNA molecular hybridization. Restricted DNA has been probed by three subclones of wheat nuclear ribosomal DNA covering the entire repeat unit. No significant difference was detected in the extent of methylation of ribosomal DNA of the doubled haploid lines with respect to the parental line. On the other hand, a variation has been found in the organization of the nontranscribed spacer region of ribosomal DNA of the first cycle doubled haploid line. This variation remains stable after a second cycle of in vitro androgenesis. However, one out of five second cycle doubled haploid lines so far tested showed an additional hybridization band present in the parental line but lacking in the first cycle doubled haploid line.

A comparison between mitochondrial DNA of an isogenic male-sterile (5) and male-fertile (F) couple (HA89) of sunflower.

Mitochondrial DNA from etiolated seedling of male-fertile (F) and male-sterile (S) sunflower (Helianthus annuus L.) has been investigated for the first time in an isogenic couple HA89. 1. The mt-DNAs isolated from the S and F cytoplasms give a sharp and symmetrical band at 1.706 g/ml in an analytical CsCI gradient ultracentrifugation. 2. In polyacrylamide gel it is found that mitochondria from F cytoplasm contain a Low-Molecular-Weight (LMW) mt-DNA molecule of 1.45 Kilobase-pairs (kb) in addition to the major mt-DNA. On the contrary, mitochondria from S cytoplasm contain no LMW molecule. Treatments with DNAse, RNAse and Nuclease S1 show that this LMW mt-DNA molecule consists of a supercoiled circular DNA. The length and form of the LMW molecule are confirmed by electron microscopy observations. 3. The mt-DNAs from S and F cytoplasms have also been investigated using restriction endonuclease digestions (Sa11, Xho1, Bg11). The electrophoresis of the resulting fragments reveals several differences between the S and F cytoplasm. It is not known whether the differences observed between F and S mt-DNA are correlated with the cytoplasmic male sterility phenotype.

An internal part of the chloroplast atpA gene sequence is present in the mitochondrial genome of Triticum aestivum: molecular organisation and evolutionary aspects.

An internal part of the chloroplast atpA gene has been identified in the mitochondrial DNA of Triticum aestivum. It is located near the 18S-5S ribosomal genes and partially contained within a repeated sequence. Comparison of the transferred sequence with the original ct sequence reveals several nucleotide changes and shows that neither 5' nor 3' ends are present in the mt genome. No transcript of this region could be detected by Northern analysis. This sequence is present in mitochondrial genomes of other tetraploid and diploid species of Triticum, also in the vicinity of the 18S-5S ribosomal genes, suggesting a unique transfer event. The date of this event is discussed.

T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells.

The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.