Three-point Cross Research Articles

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. We have demonstrated the feasibility of this fine-scale mapping approach in N. crassa by refining the location of r(Sk-2).

The misuse of the three-point cross for estimating chiasma interference

The building of genetic maps in diploid organisms by crosses between different genotypes and estimation of recombination frequencies from the obtained segregation data has been successfully used since a very young step in the birth of genetics. The three-point cross methodology has facilitated this task and has demonstrated at the same time that genetic distances are not additive, as some recombinant products are not recognised in the progeny. Three-point cross also allows to examine if chiasma interference exists and its evaluation. Here I show that the classical method of this estimation is erroneous and inevitably determines the apparition of a spurious, positive interference, which has been claimed to be an almost general phenomenon. Interference can only be estimated from a precise knowledge of the number of crossing over events occurring in meiocytes.

Bm kettin, homologue of the Drosophila kettin gene, is located on the Z chromosome in Bombyx mori and is not dosage compensated.

In Bombyx mori, the female is the heterogametic sex and the sex determining system is referred to as ZZ/ZW. In a previous study, we found that this insect does not show dosage compensation at the transcriptional level. To confirm the validity of our conclusion, we investigated whether or not another sex-linked gene is dosage compensated. To identify new Z-linked genes, total RNA from reciprocal hybrid females between the silkworm strains p50 and C108 was compared using the differential display technique. Nine cDNA fragments corresponding to several differentially expressed mRNAs were cloned and sequenced. The analysis of nucleotide sequence polymorphisms confirmed that one of these cDNAs, ZDD4, originated from the Z chromosome. The amino acid sequence deduced from ZDD4 has homology with kettin, a modular protein in the Z-disc of Drosophila melanogaster muscles. On immunoblots of Bombyx larval muscle proteins a polypeptide of 380 kDa was labelled with antibody to the ZDD4 peptide. We considered that the gene corresponding to ZDD4 encodes a kettin homologue in the silkworm, and denote it as Bm kettin. By a three-point cross, Bm kettin was mapped at 40.0 CM on the Z chromosome. Southern blot analysis revealed that Bm kettin was present at one copy in the genome. Northern blot analysis showed that Bm Kettin mRNA was 9.1kb in length, and that the level of the mRNA in males was two times greater than that of females. Taken together with our previous observations, the present data suggest that lack of dosage compensation is a general rule in B. mori. Moreover, the twofold difference in Bm kettin expression between males and females may help explain the sexual difference in the wing flapping activity observed in some groups of Lepidoptera.

Isolation of cDNAs for mouse phenol and bilirubin UDP-glucuronosyltransferases and mapping of the mouse gene for phenol UDP-glucuronosyltransferase (Ugtla1) to chromosome 1 by restriction fragment length variations.

The mouse gene for phenol UDP-glucuronosyltransferase (UDPGT; Ugtla1) was mapped at 42 cM on chromosome 1, a position identical to that of the gene for bilirubin UDPGT (Ugtla1), from linkage analysis of a three-point cross test with Idh-1, En-1, and Ugtla1 as marker genes. The cDNAs for mouse phenol and bilirubin UDPGTs, isolated after amplification by PCR, shared an identical 3'-half region. Our results strongly suggest that mouse bilirubin and phenol UDPGTs are expressed from a single gene and involve alternative splicing events. We also detected duplication of the gene for phenol UDPGT in all mouse strains examined with the exception of MOL-MIT and SUB-SHH.

Genetics of reproductive isolation in the Drosophila simulans clade: DNA marker-assisted mapping and characterization of a hybrid-male sterility gene, Odysseus (Ods).

In this study, we address the question of whether there exist major genes that cause complete male sterility in the interspecific hybrids of Drosophila and, if they do, how these genes may be characterized at the molecular level. Our approach is to introgress small segments of the X chromosome from Drosophila mauritiana (or Drosophila sechellia) into Drosophila simulans by repeated backcrosses for more than 20 generations. The introgressions are monitored by both visible mutations and a series of DNA markers. We compare the extent of introgressions that cause male sterility with those that do not. If a major sterility factor exists, there should be a sharp boundary between these two classes of introgressions and their breakpoints should demarcate such a gene. Furthermore, if male sterility is the only major fitness effect associated with the introgression, recombination analysis should yield a pattern predicted by the classical three-point cross. Both the genetic and molecular analyses suggest the presence of a major sterility factor from D. mauritiana, which we named Odysseus (Ods), in the cytological interval of 16D. We thus formalize three criteria for inferring the existence of a major gene within an introgression: (1) complete penetrance of sterility, (2) complementarity in recombination analysis, and (3) physical demarcation. Introgressions of Ods from D. sechellia do not cause sterility. Twenty-two introgressions in our collection have breakpoints in this interval of about 500 kb, making it possible to delineate Ods more precisely for molecular identification. The recombination analysis also reveals the complexity of the introgressed segments--even relatively short ones may contain a second male sterility factor and partial viability genes and may also interfere with crossovers. The spermatogenic defects associated with Ods and/or a second factor were characterized by phase-contrast microscopy.

Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene

The mouse cDNA encoding DNA topoisomerase I (Topol) was cloned and the nucleotide sequence of 3512 bp was determined. The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart. Overall aa sequence homology between the mouse and human, and between the mouse and yeast ( Saccharomyces cerevisiae) sequences was 96% and 42%, respectively. The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Gcg, Ada, and a as marker genes.

Mapping of the mouse bilirubin UDP-glucuronosyltransferase gene (Gnt-1) to chromosome 1 by restriction fragment length variations.

Restriction endonuclease fragment length variations (RFLVs) were detected by using a rat cDNA probe for the bilirubin UDP-glucuronosyltransferase (UDPGT) gene between two mouse strains, 129/Sv and MOL-MIT. RFLVs of the gene were found by EcoRI and PvuII digestions. From linkage analyses of the three-point cross test using Elo and En-1 as marker genes, the bilirubin UDPGT gene was mapped at position 37 on chromosome 1. Bilirubin and phenol UDPGTs have been suggested to be expressed by a single gene by alternative splicing in human and rat. The mouse bilirubin UDPGT gene was named Gnt-1.

Assignment of Two Enzyme Loci to the X Chromosome of Anopheles quadrimaculatus Species A

Analysis of isozyme variability in four natural populations of Anopheles quadrimaculatus Species A indicated that the loci for Malic enzyme (Me) and Mannose phosphate isomerase-1 (Mpi-1) are on the X chromosome. There were female heterozygotes, but no male heterozygotes were observed. Strains fixed for fast- and slow-migrating allozymes were devised and crossed. Progeny phenotypes conformed to expectations for sex linkage; female progeny were heterozygous, and male progeny were hemizygous for the maternal allele. The three-point cross, using the Me and Mpi-1 loci with the sex-linked mutant rose eye (ro), established the gene sequence Mpi-1-11.1-Me-40.8-ro.

Biochemical and genetic characterization of esterase-27 (ES-27), the major plasma cholinesterase of the house mouse (Mus musculus)

Esterase-27A (ES-27A) was characterized in strain A/WySnA by a cascade of seven bands seen after disc electrophoresis of serum and subsequent staining for esterase. ES-27A catalyses the hydrolysis of thiocholine butyrate and is strongly inhibited by 100 microM tetraisopropyl pyrophosphamide (isoOMPA). Hence, the enzyme was concluded to be a cholinesterase EC 3.1.1.8. A heat-labile form termed ES-27B was represented by strain AKR/Han. From a three-point cross (AKR/Han, A/Wy) and a five-point cross (AKR/Han, SEG/1), the gene order on chromosome 3 was concluded to be centromere-Car-2-Es-26-Es-27-Amy-1-Adh-1.

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probe Hox-3.1 for the homeo box-3.1 gene and also the c-myc oncogene probe for exon 2. RFLV of Hox-3.1 were found in HindIII restriction patterns, and RFLV of the Myc-1.2 gene in EcoRV patterns. From the RFLV, the Hox-3.1 and Myc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% between Myc-1.2 and Gpt-1, 30.2% between Gpt-1 and Gdc-1, and 9.4% between Gdc-1 and Hox-3.1. The following order of these genes is proposed, Myc-1.2--Gpt-1--Gdc-1--Hox-3.1. All laboratory strains carry the Hox-3.1a and Myc-1.2a alleles. Among strains of wild origin, domesticus strains carry only the Hox-3.1a and Myc-1.2a alleles, as do the laboratory strains. One strain of brevirostris carries the Hox-3.1a and Myc-1.2b alleles. Other wild subspecies from Europe and Asia, M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai carry the Hox-3.1b and Myc-1.2b alleles.

Restriction fragment length variations and chromosome mapping of two mouse metallothionein genes, Mt-1 and Mt-2.

Restriction endonuclease fragment length variations (RFLVs) were found through the use of cDNA probes for metallothionein genes 1 (Mt-1) and 2 (Mt-2) in the mouse. RFLVs were detected in restriction patterns generated by BglII and XbaI in the Mt-1 gene and by PvuII in the Mt-2 gene. All laboratory strains carry the Mt-1a and Mt-2a alleles. Among strains of wild origin, some Western European subspecies (Mus mus domesticus and M. m. brevirostris) also carry the Mt-1a and Mt-2a alleles. In contrast, a European subspecies (M. m. musculus) and the great majority of subspecies from East Asian countries (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Mt-1b and Mt-2b alleles. A domesticus strain from Bulgaria and two castaneus strains from Thailand and Philippines carry the intermediate combination of Mt-1b and Mt-2a alleles. Using the RFLVs, we mapped the Mt-1 and Mt-2 genes on chromosome 8, and they appear to be very closely linked since no recombination was observed between them in any of the mice examined. Data from three-point cross tests showed that the recombination frequencies are 4.31% between Os and Mt, 15.52% between Mt and Prt-2, and 19.83% between Os and Prt-2. The gene order of Os-Mt-1,Mt-2-Prt-2 has been confirmed.

Organic aciduria and butyryl CoA dehydrogenase deficiency in BALB/cByJ mice.

A metabolic screening program of inbred strains of mice has detected a marked organic aciduria in the BALB/cByJ strain. Gas chromatographic and mass spectrometric analysis identified large quantities of n-butyrylglycine plus lesser quantities of ethylmalonic acid. Crosses with the nonexcreting C57BL/6J strain indicate that this condition is inherited as an autosomal recessive trait. Independently from this screening a variant with no detectable enzyme activity of butyryl CoA dehydrogenase (BCD) in liver and kidney of the BALB/cByJ strain but not other BALB/c sublines was discovered. Data from a three-point cross indicated that the null variant maps to the structural locus for the enzyme, Bcd-1, on chromosome 5. The findings indicate that a mutation at or near Bcd-1 in the BALB/cByJ strain resulted in a biochemical abnormality manifest as the BCD deficiency. It is concluded that accumulation of butyryl CoA due to a block in the oxidation of short-chain fatty acids results in an overproduction of organic metabolites leading to the observed organic aciduria. The fact that other BALB/c substrains do not exhibit this abnormality further suggests that this disorder reflects subline divergence within the BALB/c family.

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.

Linkage analysis of the murine interferon alpha locus (Ifa) on chromosome 4.

We performed linkage analysis of the murine interferon alpha gene cluster, Ifa, with three different marker genes on chromosome 4: the major urinary protein locus (Mup-1), the brown locus (b) and the misty locus (m). The gene order (from centromere) with intervening percent recombination derived from a first three-point cross is Mup-1--13.6 (+/- 3.6)--Ifa--7.9 (+/- 2.8)--m and from a second three-point cross Mup-1--1.7 (+/- 1.7)--b--3.5 (+/- 2.4)--Ifa. The combined results indicate that the gene order, from the centromere, is Mup-1--b--Ifa--m.

Ly-33: a new lymphocyte alloantigen controlled by a gene linked to Ly-17 locus on mouse chromosome 1.

Several lymphocyte surface antigens are encoded by genes in the Mls region on mouse chromosome 1 [Mls, Festenstein et al. 1977; Ly-9, Ledbetter et al. 1979, Hogarth et al. 1980, Mathieson et al. 1980; Ly-17 (Ly-m20), Shen and Boyse 1980, Kimura et al. 1981; Ly-22, Tada et al. 1983]. In this report we describe a new Ly antigen (Ly-33) which is controlled by a gene linked to the Ly-17 locus on chromosome 1. A hybridoma line, N19.310, was obtained by the fusion of NS-1 myeloma cells with spleen cells from a GR/A mouse which was hyperimmunized with ASL-1, an A strain-derived T-cell tumor. N19.310 secretes IgM(K) immunoglobulin as determined by immunodiffusion (Ouchterlony and Nilsson 1978). Tissue distribution of the Ly-33 alloantigen was determined by using the direct cytotoxicity assay previously described by Kimura and coworkers (1980). With rabbit serum as a complement source, the antibody lysed 90%, 100%, 100%, and 70% of celts in thymus, lymph node, spleen, and bone marrow, respectively. The expression of the Ly-33 alloantigen on nonlymphoid tissues was tested by cytotoxicity absorption assay using the homogenates obtained from brain, liver, and kidney. They all absorbed the cytotoxic activity of Ly-33-specific antibody (data not shown). The strain distribution of the Ly-33 alloantigen was assessed by testing lymph node cells of a panel of inbred strains including CXS recombinant inbred strains, derived from the cross between BALB/c and STS/A (Michalides et al. 1985). The result is summarized in Table 1. The strain distribution of Ly-33 in CXS RI strains suggests a linkage between Ly-33 and Ly-17, as only three recombinants are found among 14 strains (Michalides et al. 1985; J. Hilgers, personal communication). To confirm the linkage and determine the position of Ly-33 on chromosome 1, a three-point cross experiment was performed by using Ly-22, Ly-17, and Ly-33 loci in (BALB.B x GR/A)F 1 x GR/A backcross mice. As shown in Table 2, among 135 backcross mice, 33, 27, and 8 recombinants between Ly-33 and

Linkage of diabetes insipidus and agouti genes in the rat.

Linkage of the hooded (h), agouti (A), and diabetes insipidus (di) genes was found in (ACI X DI)F1 X DI backcross rats. The genetic map distance A-di for females and for males was 19 +/- 5 and 28 +/- 5 cM, respectively. However, this difference was not significant. The combined data showed the map distance to be 25 +/- 4 cM. The three-point cross showed the following corrected distances and order of genes: h-42 +/- 4-A-25 +/- 4-di. However, the linkage of h and A, although significant (chi 2 = 9.03, P less than 0.001), is only tentative and must be confirmed by additional studies.

Genetic analysis of the Nd-s mutation in the silkworm, Bombyx mori.

To investigate the distance between Nd-s and Fib-L on the 14th chromosome of Bombyx mori, a three-point test cross involving U, Nd-s, and Fib-L was carried out. The crossover frequency between U and Fib-L or U and Nd-s was 24.6%. Crossingover between Fib-L and Nd-s was not observed. These results indicate that Fib-L is closely linked with Nd-s. In the fibroin slightly secreted into the lumen of the posterior silk gland of the Nd-s mutant silkworm, fibroin L-chain was not detectable. A possibility that the Nd-s phenotype is caused by a mutation in the L-chain gene, is discussed.

Genetic analysis of transposon-induced mutations of the Rac prophage in Escherichia coli K-12 which affect expression and function of recE.

Fourteen mitomycin-resistant revertants of a recB21 recC22 strain were isolated after Tn5 mutagenesis. Eight of the mutations (type I) were essentially inseparable from aphA+ (Kanr) of Tn5; six (type II) were not. We hypothesize that the former are Tn5 and that the latter are IS50 insertions. Because of their phenotypic similarity to sbcA and sbcB mutations, which also suppress recB21 recC22, we have called them sbc mutations. sbc-lll::Tn5 was cotransducible with nirR and has thereby been located at position 29.8 on the Escherichia coli map in the vicinity of the Rac prophage and sbcA mutations. A recB21 recC22 sbc-lll::Tn5 strain was subjected to Tn10 mutagenesis, and a mitomycin- and UV-sensitive mutant was isolated. tet+ of Tn10 was 85% cotransducible with aphA+ of Tn5, locating these two transposons 0.1 map unit apart. A three-point cross located the Tn10 mutation at position 29.7. We hypothesize that the Tn10 insertion is located in recE and that the Tn5 and IS50 insertions activate expression of this gene. sbc-lll::Tn5 was found to be cis acting and dominant to its wild-type allele as were two sbcA mutations (sbcA1 and sbcA6). Five other type I and type II insertion mutations were dominant to their wild-type alleles. We hypothesize that the sbc insertion and sbcA mutations affect transcription regulation of recE and discuss the possibility that they do so differently.

Linkage of the structural gene for uroporphyrinogen I synthase to markers on mouse chromosome 9 in a cross between feral and inbred mice.

The Ups locus has been mapped to mouse chromosome 9 in a three-point cross. The observed gene order is centromere-Ups-15-Mpi-1-22-Mod-1. Ups is unlinked to Lv, which encodes the previous enzyme in the heme biosynthesis pathway. Feral mice collected at Skive, Denmark, have been characterized at several biochemical loci; multiple differences from inbred strains make this a useful stock for linkage analysis.

GENETIC CONTROL AND LINKAGE RELATIONSHIPS AMONG AMINOPEPTIDASES IN MAIZE

Maize aminopeptidase is coded by four genes. Amp1 and Amp2 have been localized to chromosome 1. A three-point cross shows the gene order to be Amp2-15%-Amp1-33%-Adh2 (alcohol dehydrogenase). Amp3 and Amp4 assort independently of each other and of chromosome 1 aminopeptidases. Another linkage relationship among the maize genes Amy2 (amylase), Cat1 (catalase), and Amp3 exists, but the chromosome location has yet to be established unequivocally.