Restriction Fragment Length Polymorphism Mapping Research Articles

Molecular Cloning of a Neurospora crassa Carotenoid Biosynthetic Gene (Albino-3) Regulated by Blue Light and the Products of the White Collar Genes

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.

Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14.

We describe a general strategy for the genetic mapping in parallel of multiple restriction fragment length polymorphism (RFLP) loci. This approach allows the systematic identification for cloning of physical genetic loci within about 100 kb of any gene in Caenorhabditis elegans. We have used this strategy of parallel RFLP mapping to clone the heterochronic gene lin-14, which controls the timing and sequence of many C. elegans postembryonic developmental events. We found that of about 400 polymorphic loci in the C. elegans genome associated with the Tc1 family of repetitive elements, six are within 0.3 map unit of lin-14. The three closest lin-14-linked Tc1-containing restriction fragments were cloned and used to identify by hybridization an 830-kb region of contiguous cloned DNA fragments assembled from cosmid and yeast artificial chromosome libraries. A lin-14 intragenic recombinant that separated a previously cryptic lin-14 semidominant mutation from a cis-acting lin-14 suppressor mutation was used to map the location of the lin-14 gene to a 25-kb region of this 830-kb contig. DNA probes from this region detected lin-14 allele-specific DNA alterations and a lin-14 mRNA. Two lin-14 semi-dominant alleles, which cause temporally inappropriate lin-14 gene activity and lead to the reiterated expression of specific early developmental events, were shown to delete sequences from the lin-14 gene and mRNA. These deletions may define cis-acting sequences responsible for the temporal regulation of lin-14.

Restriction fragment length polymorphism maps and the concept of graphical genotypes

With the advent of high density restriction fragment length polymorphism (RFLP) maps, it has become possible to determine the genotype of an individual at many genetic loci simultaneously. Often, such RFLP data are expressed as long strings of numbers or letters indicating the genotype for each locus analyzed. In this form, RFLP data can be difficult to interpret or utilize without complex statistical analysis. By contrast, numerical genotype data can also be expressed in a more useful, graphical form, known as a "graphical genotype", which is described in detail in this paper. Ideally, a graphical genotype portrays the parental origin and allelic composition throughout the entire genome, yet is simple to comprehend and utilize. In order to demonstrate the usefulness of this concept, graphical genotypes for individuals from backcross and F2 populations in tomato are described. The concept can also be utilized in more complex mating schemes involving two or more parents. A model that predicts the accuracy of graphical genotypes is presented for hypothetical RFLP maps of varying marker spacing. This model indicates that graphical genotypes can be more than 99% correct in describing a genome of total size, 1000 cM, with RFLP markers located every 10 cM. In order to facilitate the application of graphical genotypes to genetics and breeding, we have developed computer software that generates and manipulates graphical genotypes. The concept of graphical genotypes should be useful in whole genome selection for polygenic traits in plant and animal breeding programs and in the diagnosis of heterogenously based genetic diseases in humans.

Association of class II DNA restriction fragments with responsiveness to Ambrosia artemisiifolia (short ragweed)-pollen allergen Amba V in ragweed-allergic patients

Human IgE and IgG antibody responsiveness to the short ragweed-pollen allergen Amb a V (formerly known as Ra5) has been found to be strongly associated with HLA-D specificities Dw2 and DR2 in ragweed-allergic white individuals. To study the molecular basis of these associations, restriction fragment length polymorphism (RFLP) mapping was performed on a group of 45 white ragweed-allergic patients with full-length HLA-DRβ, DQβ, and DQα cDNA probes. The data on 41 of these subjects were used for the purposes of statistical analysis. With the DRβ probe, we found that the presence of three polymorphic restriction fragments correlated with responsiveness to Amb a V and with the DR2 specificity, namely, a 6.5 kb Eco RI fragment, a 9.4 kb Hind III fragment, and a 2.2 kb Hind III fragment. The presence of four fragments detected with the DQβ probe correlated with responsiveness to Amb a V and with Dw2 specificity: a 2.3 kb Eco RI fragment, a 13.0 kb Pst I fragment, a 2.9 kb Taq I fragment, and a 5.2 kb Eco RV fragment. The DRβ Eco RI 6.5 kb and the DQβ Eco RI 2.3 kb fragments were studied in detail; the concordant presence of these fragments was even more strongly associated with responsiveness to Amb a V. Fifteen of 17 responders had both fragments, whereas only one of 24 nonresponders had both fragments ( p = 5 × 10 −7). This is the first time that such an association has been found between a person's immune response to a well-defined antigen and a set of HLA class II DNA restriction fragments.

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene, Hox-2.1.

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments from HindIII digestion. The great majority of laboratory strains of mice carries the Hox-2.1a allele. Only two laboratory strains carry the Hox-2.1b allele. Among strains of wild origin, the European subspecies (Mus m. domesticus, M. m. brevirostris, and M. m. musculus) and some Asian subspecies (M. m. castaneus) carry the Hox-2.1a allele. The subspecies from Far Eastern countries (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Hox-2.1b allele. Using the RFLP, the Hox-2.1 gene was mapped on chromosome 11. Three-point cross test data showed that the recombination frequency is 29.6% between the Hba and the Hox-2.1 genes and 23.5% between the Hox-2.1 and the Es-3 genes. The gene order of Hba-Hox-2.1-Es-3 has been confirmed.

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene,Hox-2.1

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene,Hox-2.1

Restriction fragment length polymorphism linkage map for Arabidopsis thaliana.

We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; greater than 50% of the genome is within 1.9 centimorgans, or approximately 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers in two different crosses. The restriction fragment length polymorphism linkage groups were integrated with the five classically mapped linkage groups by virtue of mapped mutations included in these crosses. Markers consist of both cloned Arabidopsis genes and random low-copy-number genomic DNA clones that are able to detect polymorphisms with the restriction enzymes EcoRI, Bgl II, and/or Xba I. These cloned markers can serve as starting points for chromosome walking, allowing for the isolation of Arabidopsis genes of known map location. The restriction fragment length polymorphism map also can associate clones of unknown gene function with mutant phenotypes, and vice versa.

Extensive restriction fragment length polymorphisms in a new isolate of Chlamydomonas reinhardtii.

A new field isolate of the unicellular green alga Chlamydomonas reinhardtii with useful properties for restriction fragment length polymorphism mapping is described in this report. The isolate, S1-D2 (mating type -), was the only strain found among 24 Chlamydomonas isolates taken from many locations which was interfertile with laboratory strains of C. reinhardtii. It mates at high efficiency, giving tetrads with excellent viability. Using cloned probes for both nuclear and chloroplast genes, we have found numerous restriction fragment length polymorphisms between S1-D2 and laboratory strains of C. reinhardtii.

Structural diversity of the patatin gene family in potato cv. Desiree.

We have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L. cv. Desiree (2n = 4x = 48). Nine dihaploid derivatives (2n = 2x = 24) of cv. Desiree were isolated by gynogenesis through prickle pollination with S. phureja Juz et Buk. Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases HindIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage lambda replacement vector EMBL4. This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity. Most of the 64-72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered. Four of the 6-8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass. Of the isolated clones, 4 contained apparent pseudogenes lacking 5' control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5' region due to the cloning event. The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5' coding and non-coding DNA (from 77%-95%). In one case the extent of homology differed, with complete sequence divergence upstream of position -80 from the start of transcription. The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv. Desiree as a host for potato transformation experiments.

Cloning of mtr, an amino acid transport gene of Neurospora crassa.

Translocation of neutral aliphatic and aromatic amino acids across the plasma membrane of the ascomycete Neurospora crassa requires a functional gene product of the mtr locus. Mutations at this locus are defective in transport of those amino acids. We have cloned the mtr+ gene of Neurospora crassa from an ordered cosmid library of genomic DNA and produced a preliminary restriction map of 2.9 kilobases of genomic DNA that encompasses the mtr coding region. We have confirmed that the cloned DNA regions contain the mtr gene sequence by restriction fragment length polymorphism mapping and have determined that the cloned sequence codes for a messenger RNA transcript of approximately 1200 nucleotides in length.

Therapeutic and genetic prospects of an atypical affective disorder.

The utility of bipolar type II affective disorder subgrouping is discussed. There is low diagnostic agreement among clinicians for this putative condition. However, the clustering of cases in families and the poor response to standard treatments suggest that it is a distinct subgroup. The clinical features of the depressive phase of this condition including chronicity, intermittency, hyperphagia, hypersomnia, and reactivity relate it to the constructs of "hysteroid dysphoria," atypical depression, and seasonal affective disorder. Its association to several abnormal motivated behaviors such as alcoholism and eating disorders allows the speculation that a distinct morbid mechanism involving serotonin may underlie it and that new serotonin reuptake blocking drugs may be useful in treating it. Finally, the genetic identity of this subgroup in all likelihood will be established or rejected by genetic linkage studies utilizing the restriction fragment length polymorphism map of the genome.

The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize

Two differentially expressed genes encode isoenzymes of sucrose synthase in Zea mays. A clone of the shrunken 1 (Sh1) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (Css), the structural gene for the isoenzyme expressed in embryo and other tissues. The Css clone was positively identified by RNA blot analysis of RNA from wild type and a sh1 deletion stock and by analysis of the in vitro translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an r-x1 stock, coupled with restriction fragment length polymorphism mapping, placed the Css gene 32 map units from Sh1 on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both Sh1 and Css in root tissue is enhanced by anaerobic conditions, although Css is induced to a lesser extent than is Sh1. Thus, Css appears to be expressed constitutively, whereas Sh1 is expressed at high levels only in response to specific developmental and environmental stimuli.

Efficient cloning of genes of Neurospora crassa

We have constructed a genomic library of Neurospora crassa DNA in a cosmid vector that contains the dominant selectable marker for benomyl resistance. The library is arranged to permit the rapid cloning of Neurospora genes by either sib-selection or colony-hybridization protocols. Detailed procedures for the uses of the library are described. By use of these procedures, a modest number of unrelated genes have been isolated. The cloning of trp-3, the structural gene for the multifunctional enzyme tryptophan synthetase (tryptophan synthase, EC 4.2.1.20), is reported in detail; its identity was verified by restriction fragment length polymorphism mapping. The strategies described in this paper should be of use in the cloning of any gene of Neurospora, as well as genes of other lower eukaryotes.

Restriction fragment length polymorphism markers in relation to quantitative characters

A simple consideration of the potential of restriction fragment length polymorphism mapping, as a method of analysing the inheritance of quantitative characters, suggests that there are severe limitations to the utility of this approach.

Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa

We used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of Neurospora crassa. Restriction fragment length polymorphism mapping indicated that the cosmid DNA insert was derived from linkage group IL, between 5S rDNA locus 12 and mt, the region of the N. crassa genome that contains nit-2. We conclude that the cosmid carries the nit-2 gene.