Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters

Recombinant progeny lines of Cryptosporidium parvum were generated by coinfecting immunosuppressed mice with two genetically distinct isolates of C. parvum. Progeny lines were obtained from a cross of parental lines MD x TU114 through targeted propagation in mice of progeny oocysts originating from populations lacking one parental allele at one or more loci. For each infection lineage this process was repeated until only a single allele remained for each marker, indicating that the progeny line was clonal. To study genetic recombination, 16 progeny clones were genotyped at 40 loci located on each of the eight chromosomes. The inheritance of parental alleles was significantly skewed towards the more virulent parent isolate MD. A contiguous 476 kb segment of chromosome V displayed MD allele in all progeny recovered, while MD and TU114 alleles were detected at other loci throughout the genome. The absence of alleles from one parental isolate in this chromosomal region may indicate phenotypic selection for the MD allele during the generation of these lines. A range for the meiotic crossover frequency was determined on the basis of 40 markers and the number of meioses estimated to have taken place during the crossing experiment. C. parvum exhibits a high rate of recombination commensurate with other Apicomplexa.     

MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families

Genetic variation in the major histocompatibility complex (MHC) class IIB was tested in Japanese flounder (Paralichthys olivaceus) for survival after challenge with bacterial infection. The material consisted of 6000 Japanese flounder from 60 families challenged with Vibrio anguillarum, which causes significantly different mortality in flounder families. Five individuals from each of six high-resistance (HR) and six low-resistance (LR) families were screened for their MHC class IIB genotypes using sequence analysis. High polymorphism of MHC IIB gene and at least three loci were discovered in Japanese flounder and the rate of d(N) occurred at a significantly higher frequency than that of d(S) in PBR. Among 60 individuals, 76 alleles were discovered and 15 alleles were used to study associations between alleles and resistance to disease. We found highly significant associations between resistance towards infectious disease caused by V. anguillarum and MHC class IIB polymorphism in Japanese flounder. Some alleles appeared in both HR and LR families, while some alleles were only discovered in HR or LR families. One allele, Paol-DAB*4301, was significantly more prevalent in HR families than in LR families (P=0.023). Paol-DAB*0601, Paol-DAB*0801, Paol-DAB*2001, Paol-DAB*3803 were discovered in two HR families with high frequency. One allele, Paol-DAB*1601, was discovered in three LR families. The steady heredity of MHC class IIB alleles was observed, and the family having Paol-DAB*4301 alleles was confirmed with higher resistance to V. anguillarum. This study confirmed the association between alleles of MHC class IIB gene and disease resistance, and also detected some alleles which might be correlated with high bacterial infection resistance. The disease resistance-related MHC markers could be used for molecular marker-assisted selective breeding in the flounder.     

Analysis of VH gene replacement events in a B cell lymphoma

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Expressed hypervariable polymorphism of apolipoprotein (a)

Elevated plasma lipoprotein (a) (LP(a] levels are an independent predictor of the development of premature atherosclerosis in humans. The LP(a) particle consists of two disulfide-linked proteins, apolipoprotein (APO) B and APO(a). The APO(a) is a highly glycosylated protein which carries the LP(a) antigen. Genetic polymorphism in the APO(a) molecule has been reported, and, depending on the sensitivity of the method used, 6-11 alleles at the APO(a) structural locus have been documented in the literature. In this investigation, we have used a high-resolution SDS-agarose electrophoresis method followed by immunoblotting to screen APO(a) polymorphism in 54 families with 130 offspring. This method identified a total of 23 different APO(a) isoforms, and their genetic basis was confirmed in families. In addition to the detectable products of 23 APO(a) alleles, the family data predict the existence of a "null" allele. Of the total 270 individuals tested, 209 (77.4%) revealed double-banded phenotypes and 61 (22.6%) revealed single-banded phenotypes. In the unrelated sample of 140 individuals, however, 114 (81.4%) and 26 (18.6%) had double- and single-banded phenotypes, respectively. When the segregation pattern of single-banded phenotypes in the unrelated sample was followed in families, only nine (6.4%) were found to be true homozygotes, and the remaining 17 (12.2%) were classified as heterozygotes for the null allele. Of the 276 possible phenotypes predicted for 23 alleles in a large population, we observed 115 (42%) phenotypes in our restricted sample. On the basis of our results from the family data, we hypothesize the existence of at least 24 alleles, including a null allele, at the APO(a) structural locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene

The yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity-related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2-1, depended on the presence of a functional SAP2-2 allele. In contrast, inactivation of SAP2-1 did not in-fluence the expression of SAP2-2. The proteinase encoded by the SAP2-2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2-1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2-1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2-2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro-moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that reduced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.     

A fine analysis of glucose-phosphate-isomerase patterns in single preimplantation mouse embryos

Mouse embryos were derived from eggs heterozygous for alleles of the dimeric enzyme glucose phosphate isomerase (Gpi-1a/Gpi-1b) that had been fertilized with sperm carrying a third allele (Gpi-1c). This particular combination makes it possible to study the activity of the paternally derived as well as the maternally derived genes, the persistence of oocyte-coded enzyme throughout early development and the possible simultaneous expression of both the paternally derived allele and the maternal message. The different isozymes present in single embryos were separated by electrophoresis. The results show that the oocyte-coded glucose phosphate isomerase is gradually replaced by embryo-coded enzyme. Expression of the paternally derived allele was first detected at the morula stage, during which the translation of the maternally derived message seemed to be either exhausted or below the detection limit of our system. Some oocyte-coded enzyme persisted until after implantation.     

Glutaric aciduria type I in the Arab and Jewish communities in Israel.

Mutation analysis was performed in eight families (16 patients) with glutaric aciduria type I (GA-I), which were all the families diagnosed in Israel in the years 1987-1994. Six families were of Moslem origin and two were non-Ashkenazi Jews. The entire coding region of the cDNA of the glutaryl-CoA dehydrogenase gene was sequenced in one patient of each family. Seven new mutations were identified in 15 of 16 mutated alleles, including six point mutations: T416I (4 alleles), G390R (1 allele), and S305L, A293T, L283P, and G1O1R (2 alleles each). In addition, a 1-bp deletion at position 1173 was identified in two alleles. These findings do not provide a molecular basis for the clinical variability in GA-I families. The occurrence of multiple novel mutations in a small geographic area may be explained by their recent onset in isolated communities with a high consanguinity rate.     

Computer simulation of family selection schemes suitable for kale (Brassica oleracea L.), involving half-sib,full-sib and selfed families

Summary Three recurrent selection schemes suitable for kale (Brassica oleracea L.), involving half-sib (HS), full-sib (FS) and selfed (S) families, were compared by computer simulation. All combinations of 6, 12 and 24 families selected, out of 120 and 240 assessed, were investigated for a range of genetical models. Selection was simulated for 20 generations from an initial allele frequency of 0.05 and for 16 generations from an initial frequency of 0.20. With an initial frequency of 0.05 there was a serious loss of desired alleles ranging from 0.31 out of 20 for the HS scheme with 24 out of 240 families selected to 9.19 for the S scheme with 6 out of 120 families selected. It was concluded that if as many as 20 cultivars were included in the initial population the selection scheme should be chosen to minimise the loss. With an initial frequency of 0.20 there were no losses with 12 and 24 families selected in the HS and FS schemes respectively, and the highest loss was 2.88 for the S scheme with 6 out of 120 families selected. It was concluded that if as few as five cultivars were included in the initial population a compromise between the initial response to selection and the loss of desired alleles should be sought. Selecting 6, 12 and 24 families for the HS, FS and S schemes respectively, resulted in average relative responses per generation of 2.28, 2.74 and 2.76, respectively for the first five generations, and losses of 0.22, 0.13 and 0.35, respectively after 16 generations. Practical considerations favour the FS scheme over the S scheme.     

Gene activation of alcohol dehydrogenase in danio hybrids

The regulation of alleles encoding the enzyme alcohol dehydrogenase (ADH) was investigated in F1Brachydanio hybrids (zebra danio female x spotted danio male) by acrylamide gel electrophoresis. Both parental species showed a single, cathodal band of species-specific ADH. During development at 26 degrees C, hybrid fry showed a preferential activation of the maternally derived Adh allele. It is suggested that the low activity of the paternally derived allele may result from an incompatibility between maternal regulatory factors and the paternal regulative element controlling gene expression.     

The genetics of self-incompatibility inLeavenworthia crassa rollins (Cruciferae)

Six plants of a self-incompatible population ofLeavenworthia çrassa were grown from seed collected in nature and cross-pollinated in all combinations. The incompatibility relationships between sibs were determined in eleven of the F₁ families. A one-locus sporophytic incompatibility system was established. None of the parents was homozygous at the S locus. At least five, and possibly all six, of the parents did not share an S allele. Only one pair of alleles was shown to have different interactions in the pollen and stigmata. The identity and expression of the S alleles were determined in six families. Eight pairs of alleles were independently expressed in both the pollen grains and the stigmata. Sixteen pairs of alleles showed dominance of one allele over the other in the pollen grains or the stigmata or both.F₁ plants of two crosses between different self-incompatible races were self-incompatible. F₁ plants of six crosses between self-incompatible and self-compatible races were self-incompatible; in five of the families, the frequency of pseudo-compatibility was higher than in the self-incompatible parent. Self-incompatible hybrids from a cross between a self-incompatible and a self-compatible population provide a method for rapidly determining allelic interactions in plants with a sporophytic incompatibility system.The research was carried out at the Biological Laboratories, Harvard University, Cambridge, Massachusetts, U.S.A.     

Genetic polymorphism of adenosine deaminase and mannose phosphate isomerase in blood of arctic foxes, Alopex lagopus

Blood samples of 70 foxes, including 10 families, have been investigated by horizontal starch gel electrophoresis for the enzymes adenosine deaminase (ADA) and mannose phosphate isomerase (MPI). The observed variation of the enzymes could be explained as a result of one locus with two codominant alleles and one with three respectively. The segregation in the families of the alleles assumed for the two loci is in accordance with this genetic model. The frequency of the two alleles at the Ada locus is about the same and the slowest anodic migrating allele at the Mpi locus is the most frequent.     

The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia.

Friedreich ataxia (FA) is associated with the expansion of a GAA trinucleotide repeat in the first intron of the X25 gene. We found both alleles expanded in 67 FA patients from 48 Italian families. Five patients from three families were compound heterozygotes with expansion on one allele and an isoleucine-->phenylalanine change at position 154 on the other one. We found neither expansions nor point mutations in three patients. The length of FA alleles ranged from 201 to 1,186 repeat units, with no overlap with the normal range, and showed a negatively skewed distribution with a peak between 800 and 1,000 repeats. The FA repeat showed meiotic instability with a median variation of 150 repeats. The lengths of both larger and smaller alleles in each patient inversely correlated with age at onset of the disorder. Smaller alleles showed the best correlation, accounting for approximately 50% of the variation of age at onset. Mean allele length was significantly higher in patients with diabetes and in those with cardiomyopathy.     

A second case of somatic triple mosaicism in the CYBB gene causing chronic granulomatous disease

The most common form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene that is carried on the X-chromosome and give rise to the X-linked form of the disease. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, the catalytic core of the superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene or, less frequently, at the intron/exon borders. In addition, there are large, often multi-gene, deletions. Four mutations have also been found in the promoter regions. In contrast, very few intronic mutations have been reported. Here we describe an unusual intronic mutation that causes CGD. The mutation is the insertion of 12 bp in intron XI, accompanied by the deletion of exon 12. Remarkably, the grandmother of this patient is chimeric, carrying a normal allele, the patient's allele, and an allele with a 4-nucleotide insertion at a site adjacent to the patient's insertion, in combination with a 1.5-kb deletion within intron XI. The patient's mother carries a normal allele and the patient's allele. We propose that an initial mutational event during the grandmother's embryogenesis has undergone unsuccessful DNA repair and has resulted in two aberrant alleles, one of which has been inherited by the patient and his mother. Remarkably, in the only two kindreds that have been examined in detail where deletions originating within introns have led to CGD, both families have contained members with triple somatic mosaicism.     

Silencing of duplicate genes: a null allele polymorphism for lactate dehydrogenase in brown trout (Salmo trutta)

A previously described isozyme polymorphism at one of two skeletal muscle LdhA loci in brown trout is due to a null allele, Ldh1(n), producing no detectable catalytic activity. Homozygotes for this allele have approximately only 56% of the LDH activity in skeletal muscle relative to homozygotes for the active allele. The remaining activity results from enzyme subunits produced by other LDH loci. The Ldh1(n) allele is common and widespread throughout brown trout populations in Sweden and is also found in populations from Ireland. The persistence of duplicate gene expression for the LdhA loci in almost all salmonid species is best explained by natural selection against individuals containing null alleles. However, there is no indication of natural selection against brown trout with the Ldh1(n) allele: We suggest that the selection against individuals containing null alleles that is apparently responsible for the persistence of duplicate LdhA loci in salmonids occurs only under certain environmental conditions.      

Genetic Analysis of Delta, a Neurogenic Gene of Drosophila melanogaster

We report here the results of a genetic analysis of the gene Delta (Dl) of Drosophila melanogaster. Dl has been mapped to the band 92A2, on the basis of two pieces of evidence: (1) this band is the common breakpoint of several chromosomal aberrations associated with Dl mutations and (2) recombination mapping of alleles of five different lethal complementation groups that are uncovered by Df( 3R)Dl(FX3) (breakpoints at 91F11; 92A3). Dl was found to map most distally of all five complementation groups. The analysis of a large number of Dl alleles demonstrates the considerable genetic and functional complexity of Dl. Three types of Dl alleles are distinguishable. Most alleles behave as amorphic or hypomorphic recessive embryonic lethal alleles, which in addition cause various defects in heterozygosity over the wild-type allele. The defects are due to haplo-insufficient expression of the locus and can be suppressed by a duplication of the wild-type allele. The second class is comprised of three alleles with antimorphic expression. The phenotype of these alleles can only be reduced, rather than suppressed, by a duplication of the wild-type allele. The third group is comprised of three visible, predominantly hypomorphic alleles with an antimorphic component of phenotypic expression. The pattern of interallelic complementation is complex. On the one hand, there is a group of hypomorphic, fully penetrant embryonic lethal alleles which complement each other. On the other hand, most alleles, including all amorphic alleles, are viable over the visible ones; alleles of antimorphic expression, however, are lethal over visible alleles. These results are compatible with a rather complex genetic organization of the Dl locus.     

Quantitative variations of red-cell cytochrome b5 reductase (NADH-methemoglobin-reductase) in the Algerian population

Summary A striking proportion of Algerian subjects was reported among patients with congenital recessive methemoglobinemia due to cytochrome b₅ reductase deficiency (Kaplan et al. 1979).A population survery was carried out in red blood cells from 1000 algerian subjects. In 16 subjects, the cytochrome b₅ reductase activity was diminished by 50%. Family studies indicated the presence of a defective allele with an overall gene frequency of 0.008. Immunologically cross-reacting material was found in red cells with low cytochrome b₅ reductase activity. Leukocytes exhibited normal levels of enzyme in some families and low levels in others suggesting that at least two different deficient alleles at the DIA₁ locus were present in the Algerian population. A higher prevalence of the deficient allele(s) was found in subjects of Kabyle origin.     

Partial deletion of the beta-globin gene: a common beta-thalassaemia allele in Asian Indians

We have examined 11 families of Asian Indian origin, who are segregating beta-thalassaemia alleles, for coupled restriction enzyme site markers. A beta-thalassaemia deletion allele, which removes over 600 base pairs, is a common cause of thalassaemia in this population. This deletion can be conveniently detected in AvaII restriction enzyme digests. Consequently AvaII digests are particularly informative in this population because both the deletion and a coupled restriction site polymorphism may be simultaneously observed.     

Sequence diversity in the amino-terminal region of the malaria-vaccine candidate serine repeat antigen in natural Plasmodium falciparum populations

The amino-terminal region of the serine repeat antigen (SERA) of Plasmodium falciparum is a major malaria-vaccine candidate. Variation in this molecule is essentially dimorphic and alleles may be grouped into the types FCR3, K1 and Honduras1. The Honduras1-type is thought to be the product of homologous recombination between FCR3 and K1 alleles. Here we have examined patterns of sequence diversity in exon II of SERA gene, which encodes most of the amino-terminal region of the antigen, in wild P. falciparum isolates from Indonesia (n=60), Myanmar (n=10) and Thailand (n=14). Among the Indonesian isolates the FCR-3 type predominated (56/60), twenty of which we characterized as novel alleles. A new K1-type allele was also found. In Myanmar, however, all isolates displayed K1-type SERA sequences, which included one new allele. The Honduras1-type was not detected in both countries. In contrast, the 14 isolates from Thailand displayed all three allelic types, with one new Honduras1-type and three new K1-type alleles. On examining the global distribution of SERA alleles by combining previously published sequence data with our results, the FCR3-type alleles predominated in Indonesia, Brazil, and Solomon Islands, but were not found in wild isolates from Myanmar and Africa. Brazil was the only area where K1-type alleles were not found. The distribution of Honduras1-type alleles seems to be mostly restricted to parasite populations from Vietnam, Thailand and Africa. In the allelic families FCR3 and K1, most diversity resulted from variation in sequence and number of octamer repeat units and of allotypes encoding the stretch of serine residues. Sequence analysis indicated that both insertions and deletions of repetitive motifs (creating variation within dimorphic allelic families) and homologous recombination between alleles belonging to different allelic families (creating Honduras1-type alleles) play a role in generating new SERA alleles. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitory antibodies, sequence variation in exon II may represent one of the parasite's immune-evasion strategies.