Search

In the table shown in the Briefing "Who leads the (Ivy) League in " 'citation impact'? " (9 Mar., p. 1183), the figures shown in the columns for "Citations" and "Citation impact" for Cornell University were incorrect. They should have been "523,878" and "16.53," respectively. The ranking was correct."

This study explores the theoretical potential of "insertional mutagenesis" (i.e., mutagenesis as a result of integration of novel DNA sequences into the germ line), as a means of cloning quantitative trait loci (QTL). The approach presented is based on a direct search for mutagenic effects of a quantitative nature, and makes no assumptions as to the nature of the loci affecting quantitative trait value. Since there are a very large number of potential insertion sites in the genome but only a limited number of target sites that can affect any particular trait, large numbers of inserts will have to be generated and screened. The effects of allelic variants at any single QTL on phenotype value are expected to be small relative to sampling variation. Thus two of three stages of replicate testing will be required for each insert in order to bring overall Type I error down to negligible proportions and yet maintain good statistical power for inserts with true effects on the quantitative traits under consideration. The overall effort involved will depend on the spectrum of mutagenic effects produced by insertional mutagenesis. This spectrum is presently unknown, but using reasonable estimates, about 10,000 inserts would have to be tested, at reasonable replicate numbers (n ≧ 30) and Type I error (α=0.01) in the first testing stage, to provide a high likelihood of detecting at least one insert with a true effect on a given quantitative trait of interest. Total offspring numbers required per true quantitative mutagenic effect detected decrease strongly with increased number of traits scored and increased number of inserts per initial transformed parent. In fact, it would appear that successful implementation of experiments of this sort will require the introduction of multiple independent inserts in the original parent individuals, by means of repeated transformation, or use of transposable elements as inserts. When biologically feasible, selfing would appear to be the method of choice for insert replication, and in all cases the experiments must be carried out in inbred lines to reduce error variation due to genetic segregation, and avoid confounding mutational effects of the insert with effects due to linkage with nearby segregating QTL. The special qualifications of Arabidopsis thaliana for studies of this sort are emphasized, and problems raised by somaclonal variation are discussed.

Four thousand Escherichia coli clones containing yeast DNA inserted into the plasmid pBR313 have been isolated. Of these, 175 clones were identified as carrying yeast transfer RNA genes. The initial analysis of the inserted transfer RNA genes via the colony hybridization technique with individual radioactive transfer RNA species is reported. The data indicate that yeast transfer RNA genes are not highly clustered, although some clustering exists. In addition, it was observed that the reiteration number of different transfer RNA genes may vary extensively.

In plant pedigree breeding programs, predictions of the likelihood of program success with respect to improvement of a given quantitative character can be made on the basis of the phenotypic difference between parental lines, 2[d], and the additive genetic component, D, of the genetic variance of the quantitative character in the F∞, inbred products of the cross between them. Estimations of D are subject to uncertainty, and as a consequence, breeding program recommendations may result in decision-making errors. The power of the North Carolina Experiment III design for estimating D with regard to such errors was considered as a function of [d] and true D, and of the size and family structure of the experiment. The optimal number of F2 families and of replicates per family for the estimation of D with high power was determined under various sets of genetic conditions. The sensitivity of the procedure to the underlying true value of D and to the interval between critical decision-making thresholds was examined. Although the North Carolina Experiment III design proved robust under a wide range of genetic conditions, large experiments are required at borderline decision-situations when the difference in mean phenotypic expression between the parental lines crossed is small, while the likelihood of an equivocal decision is high when the difference between the parental lines crossed is large.

A convenient procedure for the efficient plating of cells and protoplasts at low densities (as low as 200/ml) was developed. Feeder cells from exponentially growing Petunia suspension cell cultures are embedded in solid medium and overlaid with a cellophane membrane on which the nursed cells are plated. This method allows for the rapid transfer of cells from one set of experimental conditions to another, without the need to wash the cells. Being transparent, the membrane allows for an easy macro- as well as microscopic follow-up of both feeder and nursed cells. The method was successfully utilized for the isolation and rescue of Petunia hybrida cells resistant to 6-aza-uridine (6-aza-U), methotrexate (MTX) or the aminoglycoside G-418. Finally, the possibility of utilizing feeders from distant species has been demonstrated for tobacco mesophyll protoplasts using petunia feeder cells, suggesting that this technique could also be useful in other plant cell culture systems.

Sendai virus (SV), a fusogenic agent in animal cells, was investigated for its potential to interact and possibly fuse with the plasma membranes of plant protoplasts. Adsorption of fluorescently-labelled viral particles was observed to be dependent on the presence of concanavalin A (ConA). This interaction could not be reproduced by wheat germ agglutinin. ConA adsorption to protoplasts could be reversed by an excess of free competing sugar moieties (α-methyl-mannoside, αMM) at any time. Complexes between Sendai virions, ConA and protoplasts could be dissociated by αMM, only shortly after their formation. Prolonged incubation of these complexes prevented their dissociation upon the later addition of the competing sugar. Neither dithiothreitol nor trypsinization of the viruses, known to affect the viral fusogenic ability to animal cells, modified the described adsorption properties. This is taken to mean that irreversible binding of Sendai viral particles to protoplast plasma membranes does occur, presumably via a different mechanism from that with animal cells. This should encourage studies aimed at testing the capacity of the reconstituted Sendai envelopes to serve as delivery carriers in plant genetic engineering projects.

The development of molecular genetic markers, detecting variation at the DNA level, has opened new horizons in the genetic analyses of both man and agricultural species. Agricultural applications of “Restriction Fragment Length Polymorphisms” (RFLPs), despite their promise, are limited by the formidable costs involved in examining the large populations required. It is the purpose of this contribution to point out that the introduction of oligonucleotide probes into agricultural genetics may provide a solution to the cost problems. Furthermore, the number of detectable “Oligonucleotide Polymorphisms” (OPs) should exceed by an order of magnitude the number amenable to RFLP techniques, thus opening the gates to additional applications.

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.

By making use of pedigree information and information on marker-genotypes of the parent and F-1 individuals crossed to form an F-2 population, it is possible to carry out a linkage analysis between marker loci and loci affecting quantitative traits in a cross between segregating parent populations that are at fixation for alternative alleles at the QTL, but share the same alleles at the marker loci. For two-allele systems, depending on marker allele frequencies in the parent populations, 2-4 times as many F-2 offspring will have to be raised and scored for markers and quantitative traits in order to provide power equivalent to that obtained in a cross between fully inbred lines. Major savings in number of F-2 offspring raised can be achieved by scoring each parent pair for a large number of markers in each chromosomal region and scoring F-1 and F-2 offspring only for those markers for which the parents were homozygous for alternative alleles. For multiple allele systems, particularly when dealing with hypervariable loci, only 10%-20% additional F-2 offspring will have to be raised and scored to provide power equivalent to that obtained in a cross between inbred lines. When a resource population contains novel favorable alleles at quantitative trait loci that are not present (or rare) in a commercial population, analyses of this sort will enable the loci of interest to be identified, mapped and manipulated effectively in breeding programs.

Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 μM MTX (in wild type the LD99.9 is 0.2 μM). MTX(R) phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.

Summary Amitrole-tolerant Nicotiana debneyi calli were isolated by stepwise selection to increasing amounts of the herbicide. One resistant line was examined over a period of five years. High levels of amitrole tolerance were obtained (up to 10−3). This tolerance, at the callus level, was found to be stably maintained under selective as well as non-selective growing conditions. Calli derived from plants regenerated from the initial tolerant calli also exhibited the tolerant phenotype, although to a lesser extent, and the tolerance levels varied with growth media. The influence of amitrole on organogenesis was also followed. Sexual transmission of the resistance trait to F1 (sib) progeny does not seem to obey simple Mendelian rules. The genetic data do not support the possibility of cytoplasmic maternal inheritance.

Several methods for the visualization of genetic polymorphisms at the nucleic acid level have been developed. Such polymorphisms promise to be exceedingly numerous, and may form the basis for a number of scientific and practical applications in fisheries science. An expanded number of genetic markers should increase the statistical power of marker-based studies in population genetics, for example, improving the sensitivity of biological stock assessments and of studies assessing the impact of stocking programs upon natural populations. Utilization of such genomic markers could contribute to the rapid elaboration of piscine genomic maps and to development of markers for health- and production-related traits in fishes.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10 6 -10 7 have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.

Methods are presented for determining linkage between a marker locus and a nearby locus affecting a quantitative trait (quantitative trait locus=QTL), based on changes in the marker allele frequencies in selection lines derived from the F-2 of a cross between inbred lines, or in the "high" and "low" phenotypic classes of an F-2 or BC population. The power of such trait-based (TB) analyses was evaluated and compared with that of methods for determining linkage based on the mean quantitative trait value of marker genotypes in F-2 or BC populations [marker-based (MB) analyses]. TB analyses can be utilized for marker-QTL linkage determination in situations where the MB analysis is not applicable, including analysis of polygenic resistance traits where only a part of the population survives exposure to the Stressor and analysis of marker-allele frequency changes in selection lines. TB analyses may be a useful alternative to MB analyses when interest is centered on a single quantitative trait only and costs of scoring for markers are high compared with costs of raising and obtaining quantitative trait information on F-2 or BC individuals. In this case, a TB analysis will enable equivalent power to be obtained with fewer individuals scored for the marker, but more individuals scored for the quantitative trait. MB analyses remain the method of choice when more than one quantitative trait is to be analyzed in a given population.

A petunia cell line, 1ECB, was previously isolated by the stepwise selection procedure, for resistance to methotrexate (MTX), an antimetabolite for the enzyme dihydrofolate reductase (DHFR). Using ammonium sulfate precipitates of cell lysates of cell line 1ECB and its parental cell line (WT), it was found that the mutant has an increase of 400 fold in (3)H-MTX binding capacity and a decrease in the affinity for MTX binding, at two orders of magnitude, in comparison with the WT. In addition, the DHFR specific activity in the mutant increased only moderately (5- to 10-fold), this activity is extremely sensitive to MTX inhibition as compared to the WT. It is evident that the MTX resistance of line 1ECB results mainly from overproduction of an MTX-binding protein which differs from the WT DHFR by four biochemical criteria. This protein may serve as a trap for the excess amounts of MTX to which the cells are exposed.

Genes for yeast tRNATyr and tRNAPhe have been sequenced (Goodman, Olson and Hall, 1977; Valenzuela et al., 1978) which contain additional nucleotides (intervening sequences) within the middle of the gene that are not present in the mature tRNA. We have isolated precursors to rRNATyr and tRNAPhe from a yeast temperature-sensitive mutant (at the rna1 locus) which accumulates only certain precursor tRNAs at the nonpermissive temperature. The tRNATyr and tRNAPhe precursors were analyzed by oligonucleotide mapping; they each contain the intervening sequence and fully matured 5' and 3' termini. Furthermore, these precursors were used as substrates to search for an enzymatic activity which can remove the intervening sequences and religate the ends. We have shown that wild-type yeast contains such an activity, and that this activity specifically removes the intervening sequences to produce mature-sized RNAs.

The concept of "words" in continuous languages devoid of blanks is introduced and an operational definition of words given. With this novel concept nucleotide sequences become object for linguistic analysis. The typical word size of the nucleotide language is found to be 3 to 5 (tri- to pentamers). Different genomes have distinct vocabularies. Comparison of these vocabularies can serve as a basis for revealing functional and evolutionary relatedness of sequences.

Evidence is accumulating demonstrating the ubiquity and abundance of a new class of genetic markers, restriction fragment length polymorphisms (RFLPs). These markers should allow the genetic map of agricultural species to be saturated in the near future. This holds great promise for useful applications, including: protection of breeder's rights, and more effective means for the characterization and manipulation of individual genetic loci affecting traits of economic importance.

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA Tyr -like sequences transcribed in vitro on φ80p su 3 + bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA Tyr hybridization revealed that the in vitro -synthesized RNA contained significant amounts of tRNA Tyr ; these sequences could not, however, be detected by the direct competition method in which labelled in vitro -synthesized RNA competes with E. coli tRNA Tyr for hybridization to φ80p su 3 + DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro -transcribed tRNA Tyr -like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA Tyr from its hybrids with φ80p su 3 + DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA Tyr .

New sources of genetic polymorphisms promise significant additions to the number of useful genetic markers in agricultural plants and animals, and prompt this review of potential applications of polymorphic genetic markers in plant and animal breeding. Two major areas of application can be distinguished. The first is based on the utilization of genetic markers to determine genetic relationships. These applications include varietal identification, protection of breeder's rights, and parentage determination. The second area of application is based on the use of genetic markers to identify and map loci affecting quantitative traits, and to monitor these loci during introgression or selection programs. A variety of breeding applications based on these possibilities can be envisaged for Selfers, particularly for those species having a relatively small genome size. These applications include: (i) screening genetic resources for useful quantitative trait alleles, and introgression of chromosome segments containing these alleles from resource strain to commercial variety; (ii) development of improved pure lines out of a cross between two existing commercial varieties; and (iii) development of crosses showing increased hybrid vigor. Breeding applications in segregating populations are more limited, particularly in species with a relatively large genome size. Potential applications, however, include: (i) preliminary selection of young males in dairy cattle on the basis of evaluated chromosomes of their proven sire; (ii) genetic analysis of resource strains characterized by high values for a particular quantitative trait, and introgression of chromosome segments carrying alleles contributing to the high values from resource strain to recipient strain.

The termination factor of transcription, ϱ, isolated from Escherichia coli , was found to bind to DNA. Complex formation of ϱ with different polynucleotides was studied by means of retention on nitrocellulose filters. Native Escherichia coli DNA binds to ϱ reversibly being replaced by other native or denatured DNA's. Actinomycin-D, does not interfere with the attachment of the termination factor to native DNA. Binding of ϱ to native DNA, confers partial protection against pancreatic DNase digestion. Evidence is presented for the existence of discrete sites for the attachment of the termination factor to the DNA.

BACKGROUND: Deletions and duplications of the 16p11.2 BP4-BP5 locus are prevalent copy number variations (CNVs), highly associated with autism spectrum disorder and schizophrenia. Beyond language and global cognition, neuropsychological assessments of these two CNVs have not yet been reported. METHODS: This study investigates the relationship between the number of genomic copies at the 16p11.2 locus and cognitive domains assessed in 62 deletion carriers, 44 duplication carriers, and 71 intrafamilial control subjects. RESULTS: IQ is decreased in deletion and duplication carriers, but we demonstrate contrasting cognitive profiles in these reciprocal CNVs. Deletion carriers present with severe impairments of phonology and of inhibition skills beyond what is expected for their IQ level. In contrast, for verbal memory and phonology, the data may suggest that duplication carriers outperform intrafamilial control subjects with the same IQ level. This finding is reminiscent of special isolated skills as well as contrasting language performance observed in autism spectrum disorder. Some domains, such as visuospatial and working memory, are unaffected by the 16p11.2 locus beyond the effect of decreased IQ. Neuroimaging analyses reveal that measures of inhibition covary with neuroanatomic structures previously identified as sensitive to 16p11.2 CNVs. CONCLUSIONS: The simultaneous study of reciprocal CNVs suggests that the 16p11.2 genomic locus modulates specific cognitive skills according to the number of genomic copies. Further research is warranted to replicate these findings and elucidate the molecular mechanisms modulating these cognitive performances.

Limb-girdle muscular dystrophies (LGMDs) are a group of inherited diseases whose genetic etiology has yet to be elucidated. The autosomal recessive forms (LGMD2) constitute a genetically heterogeneous group with LGMD2A mapping to chromosome 15815.1–821.1. The gene encoding the muscle-specific calcium-activated neutral protease 3 (CANP3) large subunit is located in this region. This cysteine protease belongs to the family of intracellular calpains. Fifteen nonsense, splice site, frameshift, or missense calpain mutations cosegregate with the disease in LGMD2A families, six of which were found within La Reunion island patients. A digenic inheritance model is proposed to account for the unexpected presence of multiple independent mutations in this small inbred population. Finally, these results demonstrate an enzymatic rather than a structural protein defect causing a muscular dystrophy, a defect that may have regulatory consequences, perhaps in signal transduction.

SummaryDistal myopathy refers to a heterogeneous group of disorders in which the initial manifestations are weakness and atrophy of the hands and feet. We report a family segregating an autosomal dominant distal myopathy, with multiple affected individuals in whom vocal cord and pharyngeal weakness may accompany the distal myopathy, without involvement of the ocular muscles. To our knowledge, this pedigree displays a distinct distal myopathy with the added features of pharyngeal and vocal cord dysfunction (VCPDM) that has not been previously reported. We mapped the MPD2 gene for VCPDM to chromosome 5q within a 12-cM linkage interval between markers D5S458 and D5S1972 in a large pedigree (a maximum LOD score of 12.94 at a recombination fraction of 0 for D5S393) and combined genome screening and DNA pooling successfully adapted to fluorescent markers. This technique provides for the possibility of fully automated genome scans.

Dilated cardiomyopathy (DCM) is a heart-muscle disease characterized by ventricular dilatation and impaired heart contraction and is heterogeneous both clinically and genetically. To date, 12 candidate disease loci have been described for autosomal dominant DCM. We report the identification of a new locus on chromosome 6q12-16 in a French family with 9 individuals affected by the pure form of autosomal dominant DCM. This locus was found by using a genomewide search after exclusion of all reported disease loci and genes for DCM. The maximum pairwise LOD score was 3.52 at recombination fraction 0.0 for markers D6S1644 and D6S1694. Haplotype construction delineated a region of 16.4 cM between markers D6S1627 and D6S1716. This locus does not overlap with two other disease loci that have been described in nonpure forms of DCM and have been mapped on 6q23-24 and 6q23. The phospholamban, malic enzyme 1-soluble, and laminin-alpha4 genes were excluded as candidate genes, using single-strand conformation polymorphism or linkage analysis.

Made available in DSpace on 2016-10-10T03:52:59Z (GMT). No. of bitstreams: 5 Calpainopathy_A Survey of Mutations and Polymorphisms.pdf: 349117 bytes, checksum: 5b8ce907469e1b201d28b825113b4052 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23892 bytes, checksum: afd5dad10b1d1e6dc10c8c5d25222c7a (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 1999 Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized mainly by symmetrical and selective atrophy of the proximal limb muscles. It derives from defects in the human CAPN3 gene, which encodes the skeletal muscle–specific member of the calpain family. This report represents a compilation of the mutations and variants identified so far in this gene. To date, 97 distinct pathogenic calpain 3 mutations have been identified (4 nonsense mutations, 32 deletions/insertions, 8 splice-site mutations, and 53 missense mutations), 56 of which have not been described previously, together with 12 polymorphisms and 5 nonclassified variants. The mutations are distributed along the entire length of the CAPN3 gene. Thus far, most mutations identified represent private variants, although particular mutations have been found more frequently. Knowledge of the mutation spectrum occurring in the CAPN3 gene may contribute significantly to structure/ function and pathogenesis studies. It may also help in the design of efficient mutation-screening strategies for calpainopathies. Publicado

Recently a new class of genetic polymorphism, restriction fragment length polymorphisms (RFLPs), has been uncovered by the use of restriction endonucleases which cleave DNA molecules at specific sites and cloned DNA probes which detect specific homologous DNA fragments. RFLPs promise to be exceedingly numerous and are expected to have genetic characteristics — lack of dominance, multiple allelic forms and absence of pleiotropic effects on economic traits — of particular usefulness in breeding programs. The nature of RFLPs and the methodologies involved in their detection are described and estimated costs per polymorphism determination are derived. The anticipated costs of applying RFLPs to genome mapping are considered in terms of the number of RFLPs required for a given degree of genome coverage, the number of probe × enzyme combinations tested per polymorphism uncovered, and the total number of individuals and polymorphisms scored for mapping purposes. The anticipated costs of applying RFLPs to genetic improvement are considered in terms of the number of individuals and the number of polymorphisms per individual that are scored for the various applications. Applications considered include: varietal identification, identification and mapping of quantitative trait loci, screening genetic resource strains for useful quantitative trait alleles and their marker-assisted introgression from resource strain to commercial variety, and marker-assited early selection of recombinant inbred lines in plant pedigree breeding programs and of young sires in dairy cattle improvement programs. In most cases anticipated costs appear to be commensurate with the scientific or economic value of the application.

Little is known about the origin and function of eukaryotic introns. Application of a novel linguistic approach to the analysis of intervening sequences reveals, however, that they exhibit a specific non-random vocabulary whose major feature is the utilization of mirror- symmetrical words (“mirrorrim”). Introns also manifest a significant tendency to avoid local complementarities. Possible biological implications of the corresponding loop regions in the RNA. transcripts are discussed.

Genomic DNA of Israeli Holstein-Friesian dairy cattle were screened with a battery of 17 cloned or subcloned DNA probes in an attempt to document restriction fragment length polymorphisms at a number of genetic loci. Restriction fragment length polymorphisms were observed at the chymosin, oxytocin-neurophysin I, lutropin beta, keratin III, keratin VI, keratin VII, prolactin, and dihydrofolate reductase loci. Use of certain genomic DNA fragments as probes produced hybridization patterns indicative of satellite DNA at the respective loci. Means for distinguishing hybridizations to coding sequences for unique genes from those to satellite DNA were developed. Results of this study are discussed in terms of strategy for the systematic development of large numbers of bovine genomic polymorphisms.

Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.

Transducing phages have proved very useful in studies involving specific bacterial genes carried by the phage DNA; because of their smaller size, the DNA of these phages is highly enriched in specific bacterial genes as compared with the bacterial chromosome and may be used as template for in vitro RNA transcription. In order to study the transcription of a bacterial tRNA molecule, we have used the transducing phage φ80psup 3 + (1). The su 3 + gene carried by the φ80 phage is the structural gene which specifies a tRNATyr molecule that enables the amber codon, UAG, to be read as tyrosine (2,3). There are two types of tyrosine tRNA’s (I and II) in E.coli, differing by two nucleotides in the variable loop. The main species in E.coli cells is tRNATyr II; the minor species tRNATyr is specified by two identical genes, one of which can undergo a mutation resulting in a single base change in the anticodon region of the tRNA (su 3 + ). The two tRNATyr I genes are located near the φ80 attachment site on the bacterial chromosome and can be transduced by the φ80 bacteriophage as a single or a tandem double copy.

A 14.3 kb petunia genomic DNA fragment was isolated and found to contain a single tRNA gene coding for asparagine tRNA. The nucleotide sequence of the asparagine tRNA gene and its flanking regions has been determined. This gene does not contain intervening sequences nor the 3'-end CCA sequence of the mature tRNA and presents a similar overall sequence homology (70%) to both E. coli and mammalian asparagine tRNA. As in other eukaryotic tRNA genes the 5'-flanking region does not seem to contain any special sequence that could function as a regulatory element and the 3'-end is followed by a short cluster of T that may function as the transcription termination site.

Two DNA fragments, each containing tRNA(Arg)3 and a tRNA(Asp) gene in close conjunction, have been isolated from different genomic regions of Saccharomyces cerevisiae. Nucleotide Nucleotide sequence analysis of the gene regions revealed that in both fragments the tRNA(Arg)3 coding region is located 5'-proximal to the tRNA(Asp) coding region. They are separated by an identical spacer of 10 nucleotides. Although the 5'-flanking sequences are different in the two plasmids, some similarities are observed. To test the mode of expression of this gene configuration, we transcribed the DNA fragments in a Xenopus oocyte nuclear extract. Specific transcription of the yeast tRNA genes took place in an RNA precursor which comprised both tRNA species. We report here that the precursor RNA was processed to the mature-sized tRNA molecules, indicating the presence of an enzyme activity in the Xenopus nucleus capable of cutting a dimeric tRNA precursor. This is the first observation of a eukaryotic dimeric tRNA precursor.

With the advent of the use of restriction endonucleases to map gene sequences, molecular geneticists have encountered widespread polymorphism for the location of restriction sites among individuals of a species. This observation is not surprising considering the number of supposedly neutral mutations encountered at the protean level in recent years. The genetic heterogeneity, called restriction fragment length polymorphism (RFLP), has been used to map linked genes (1) and is a potential tool in prenatal diagnosis (2). Considerable attention has been placed on the use of RFLPs in constructing a genetic map of the human genome to establish linkages with loci determining genetic diseases (3). The purpose of this paper is to point out the possible uses of this technique in agronomic improvement.