The Molecular through Ecological Genetics of Abnormal Abdomen. III. Tissue-Specific Differential Replication of Ribosomal Genes Modulates the Abnormal Abdomen Phenotype in DROSOPHILA MERCATORUM

The abnormal abdomen (aa) syndrome in Drosophila mercatorum is controlled by two major X-linked genetic elements. We have previously shown that the major X-linked element of aa is associated with the presence of large inserts in the 28S gene of the ribosomal RNA (rDNA) genes. We show that, in polytene tissue of wild-type D. mercatorum, the uninterrupted rDNA repeats are overreplicated relative to interrupted repeats. Uninterrupted rDNA repeats are also overreplicated in polytene tissue of hybrid larval offspring from wild-type and aa parents. This overreplication of uninterrupted repeats is not observed in diploid tissues of wild-type hybrids (of wild-type and aa parents) and homozygous aa larvae or in polytene tissue of aa larvae. Furthermore, molecular analysis of an aa line that has reverted to the wild type indicates that the reversion phenomenon is associated with the ability to overreplicate uninterrupted rDNA repeats in polytene tissues. The patterns of differential replication of rDNA genes in wild-type hybrids and aa larvae of D. mercatorum offer a possible mechanism for the tissue-specific control of the aa phenotype and suggest that the molecular basis for the second X-linked genetic element of aa is involved in the control of differential replication in polytene tissues.     

The Molecular through Ecological Genetics of Abnormal Abdomen. IV. Components of Genetic Variation in a Natural Population of Drosophila Mercatorum

Natural populations of Drosophila mercatorum are polymorphic for a phenotypic syndrome known as abnormal abdomen (aa). This syndrome is characterized by a slow-down in egg-to-adult developmental time, retention of juvenile abdominal cuticle in the adult, increased early female fecundity, and decreased adult longevity. Previous studies revealed that the expression of this syndrome in females is controlled by two closely linked X chromosomal elements: the occurrence of an R1 insert in a third or more of the X-linked 28S ribosomal genes (rDNA), and the failure of replicative selection favoring uninserted 28S genes in larval polytene tissues. The expression of this syndrome in males in a laboratory stock was associated with the deletion of the rDNA normally found on the Y chromosome. In this paper we quantify the levels of genetic variation for these three components in a natural population of Drosophila mercatorum found near Kamuela, Hawaii. Extensive variation is found in the natural population for both of the X-linked components. Moreover, there is a significant association between variation in the proportion of R1 inserted 28S genes with allelic variation at the underreplication (ur) locus such that both of the necessary components for aa expression in females tend to cosegregate in the natural population. Accordingly, these two closely linked X chromosomal elements are behaving as a supergene in the natural population. Because of this association, we do not believe the R1 insert to be actively transposing to an appreciable extent. The Y chromosomes extracted from nature are also polymorphic, with 16% of the Ys lacking the Y-specific rDNA marker. The absence of this marker is significantly associated with the expression of aa in males. Hence, all three of the major genetic determinants of the abnormal abdomen syndrome are polymorphic in this natural population.     

The Molecular through Ecological Genetics of Abnormal Abdomen in Drosophila Mercatorum. V. Female Phenotypic Expression on Natural Genetic Backgrounds and in Natural Environments

The abnormal abdomen (aa) syndrome in Drosophila mercatorum depends on the presence of R1 inserts in a third or more of the X-linked 28S rDNA genes and the absence of selective underreplication of inserted repeats in polytene tissues that is controlled by an X-linked locus (ur) half a map unit from the rDNA complex. This syndrome affects both life history and morphology in the laboratory. Because abnormal morphologies are rarely encountered in nature, the purpose of this study is to see if the female life history traits are still affected under more natural genetic backgrounds and environmental conditions. Two outbred stocks were extracted from the natural population living near Kamuela, Hawaii: KaaX that has only X chromosomes with ur(aa) alleles, and K+X that has only ur(+) alleles. These two stocks have nonoverlapping distributions of insert proportions, indicating strong disequilibrium between the ur locus and the rDNA complex. The KaaX stock had almost no morphological penetrance of ur(aa), indicating that genetic background is important. KaaX expressed longer female egg-to-adult developmental times, increased early adult female fecundity, and decreased female adult longevity compared with K+X. By bagging natural rots of the cactus Opuntia megacantha near Kamuela, Hawaii, it was shown that egg-to-adult developmental time is slowed down by 0.92 days in females bearing ur(aa) alleles in nature, with no detectable slowdown in ur(aa) males. The bagged rot data also indicate that females bearing ur(aa) alleles have a strong fecundity advantage in nature under some ecological conditions but not others.     

Natural selection and ribosomal DNA in Drosophila

Natural populations of Drosophila mercatorum are variable for the number of X-linked 28S ribosomal genes bearing a 5-kilobase insert. A separate polymorphic X-linked gene controls whether 28S repeats bearing the insert are preferentially underreplicated during the formation of polytene tissue. Female flies having at least a third of their 28S genes bearing the insert and lacking the ability to preferentially underreplicate inserted repeats display the abnormal abdomen syndrome. The syndrome is characterized by retention of juvenile abdominal cuticle into the adult, a slowdown in larval developmental time, and an increase in early female fecundity. The life history traits are expressed in nature and provide a basis for strong natural selection. The abnormal abdomen syndrome should be favored whenever the adult age structure is skewed towards young individuals, and field studies confirm this prediction. The closely related species, Drosophila hydei, also bears these inserts and appears to be subject to similar selection. However, D. mercatorum responds to this selection primarily through the allelic variation that controls preferential underreplication, whereas D. hydei responds primarily through adjustment of the proportion of inserted 28S genes. This is interpreted to mean that the evolution of a multigene family arises from the interaction of population-level and DNA-level processes.     

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.     

The Molecular through Ecological Genetics of Abnormal Abdomen. II. Ribosomal DNA Polymorphism Is Associated with the Abnormal Abdomen Syndrome in DROSOPHILA MERCATORUM

Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA-containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development.     

Genetics of grain amaranths. II. The inheritance of determinance, panicle orientation, dwarfism, and embryo color in Amaranthus caudatus

The genetic control of four developmental characters was studied in Amaranthus caudatus L. Determinant panicle growth was determined by one recessive gene. Two major genes governed panicle orientation, with erect panicles incompletely dominant to drooping panicles. Additional modifier genes appeared to alter expression of panicle orientation. A single recessive gene determined dwarfism. Pleiotropy or tight linkage was responsible for abnormal growth of dwarf plants. Pink embryo color was under the control of two complementary epistatic genes with one locus determining the presence or absence of red betalain pigment and the other locus regulating the expression of pigment in developing embryos.     

Genetics of intellectual disability

Early onset intellectual disability (ID) is one of the largest unsolved problems of health care. Yet, it has received very little public attention in the past because many health care professionals do not perceive it as a health condition but as a social or educational issue. In severe ID, cytogenetically visible chromosomal abnormalities like trisomy 21 continue to be common, but since the introduction of array CGH, it is becoming clear that submicroscopic deletions and duplications are equally frequent, yet previously overlooked causes of ID. Until recently, the search for gene defects causing ID has focused on the X-chromosome. So far, >80 genes have been implicated in X-linked ID, largely owing to coordinated efforts of international consortia, and mutations in these genes account for >50% of the families with this condition. Autosomal forms, either due to dominant de novo mutations or to recessive gene defects, are presumably (far) more common than X-linked ones, and their molecular elucidation is a new challenge for research in this field. As recently shown, autosomal recessive ID (ARID) is extremely heterogeneous, and common forms are unlikely to exist. Ongoing studies into the function of ID genes are shedding more light on the pathogenesis of this disorder, and there is reason to believe that at least some genetic forms of ID may be amenable to drug treatment.     

Tx1: a transposable element from Xenopus laevis with some unusual properties.

A family of transposable genetic elements in the genome of the frog, Xenopus laevis, is described. They are designated Tx1. Transposability of the elements was deduced by characterization of a chromosomal locus which is polymorphic for the presence or absence of a Tx1 element. Nucleotide sequence analysis suggested that Tx1 elements show target site specificity, as they are inserted at the pentanucleotide TTTAA in all four cases that were examined. The elements appear to have 19-base-pair (bp) inverted terminal repeats, and they are flanked by 4-bp target duplications (TTAA), although the possibility that they do not create target site duplications is discussed. Tx1 elements have several unusual characteristics: the central portion of each element is comprised of a variable number of two types of 393-bp repeating units; the rightmost 1,000 bp of the element contains separate regions potentially capable of forming bends, left-handed Z-form DNA, and alternative stem-loop structures. Comparisons among single frogs suggest that germ line transposition is relatively infrequent and that variations in numbers of internal repeats accumulate quite slowly at any locus.     

Telomerase dysfunction and dyskeratosis congenita

Dyskeratosis congenita (DC) is a multi system bone marrow failure syndrome characterized by muco-cutaneous abnormalities and an increased predisposition to malignancy. It exhibits considerable clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. The X-linked recessive form is due to mutations in dyskerin, which is a component of both small nucleolar ribonuclear protein particles and the telomerase complex. Autosomal dominant DC is due to mutations in the RNA component of telomerase, TERC. As dyskerin and TERC are both components of the telomerase complex and all patients with DC have short telomeres it appears that the principal pathology in DC relates to telomerase dysfunction. The gene or genes involved in the recessive form of DC remain elusive, though genes whose products are required for telomere maintenance remain strong candidates. The study of DC has highlighted the critical role of telomerase and the consequences, including premature aging and malignancy, of its dysfunction.     

A new copia-like transposable element found in a Drosophila rDNA gene unit.

We have discovered a member of a new family of copia-like transposable elements inserted into the non-transcribed spacer between two ribosomal genes (rDNA). This family, which we call 3S18, consists of at least 15 elements which are scattered throughout the Drosophila melanogaster genome. The elements of this family are approximately 6.5 kb long and have 0.5 kb terminal direct repeats. All of the elements appear to have the same restriction sites. The element is mobile as the size pattern of homologous fragments varies among different strains. In situ hybridization results confirm the scattered location and transposable qualities of 3S18. The element is not transcribed into abundant RNA.     

Utility and efficiency of linked marker genes for genetic counseling. III. Proportion of informative families under linkage disequilibrium.

A marker locus closely linked to a disease locus is often useful for genetic counseling provided that a counselee is heterozygous at both disease and marker loci. Furthermore, the linkage phase of these genes in the counselee must be known. When the linkage between the disease and marker loci is very close, one often finds linkage disequilibrium between the loci. To evaluate the effect of such nonrandom associations on the utility of linked marker genes for genetic counseling, the proportion of informative families is studied for X-linked recessive and autosomal dominant diseases. This proportion is higher for X-linked genes than for autosomal genes, if other factors are the same. In general, codominant markers are more useful than dominant markers. Also, under appropriate conditions, the proportion of informative families is higher when linkage disequilibrium is present. The results obtained in this paper are useful for evaluating the utility of polymorphic restriction endonuclease cleavage sites as markers in genetic counseling.     

Medico-genetical study of the population of the Kostroma Region. IV. Genetic load and diversity of hereditary pathology in 5 districts

Data on the prevalence of hereditary diseases in five regions of the Kostroma province were obtained and analysed. 28 autosomal recessive, 25 autosomal dominant and 4 X-linked recessive disorders were found. Segregation analysis proved the rightness of the material subdivision, according to the type of inheritance. The load of hereditary diseases in five regions was: 0.86 +/- 0.09 X 10(3) for autosomal recessive, 0.97 +/- 0.1 X 10(3) for autosomal dominant and 0.36 +/- 0.09 X 10(3) for X-linked recessive disorders. The problems of prevalence of hereditary diseases connected with population structure is discussed.     

Evolution of target specificity in R1 clade non-LTR retrotransposons

Although most non-long terminal repeat (non-LTR) retrotransposons are inserted throughout the host genome, many non-LTR elements in the R1 clade are inserted into specific sites within the target sequence. Four R1 clade families have distinct target specificity: R1 and RT insert into specific sites of 28S rDNA, and TRAS and SART insert into different sites within the (TTAGG)(n) telomeric repeats. To study the evolutionary history of target specificity of R1-clade retrotransposons, we have screened extensively novel representatives of the clade from various insects by in silico and degenerate polymerase chain reaction (PCR) cloning. We found four novel sequence-specific elements; Waldo (WaldoAg1, 2, and WaldoFs1) inserts into ACAY repeats, Mino (MinoAg1) into AC repeats, R6 into another specific site of the 28S rDNA, and R7 into a specific site of the 18S rDNA. In contrast, several elements (HOPE, WISHBm1, HidaAg1, NotoAg1, KagaAg1, Ha1Fs1) lost target sequence specificity, although some of them have preferred target sequences. Phylogenetic trees based on the RT and EN domains of each element showed that (1) three rDNA-specific elements, RT, R6, and R7, diverged from Waldo; (2) the elements having similar target sequences are phylogenetically related; and (3) the target specificity in the R1 clade was obtained once and thereafter altered and lost several times independently. These data indicate that the target specificity in R1 clade retroelements has changed during evolution and is more divergent than has been speculated so far.     

OFD1 Is Mutated in X-Linked Joubert Syndrome and Interacts with LCA5-Encoded Lebercilin

We ascertained a multi-generation Malaysian family with Joubert syndrome (JS). The presence of asymptomatic obligate carrier females suggested an X-linked recessive inheritance pattern. Affected males presented with mental retardation accompanied by postaxial polydactyly and retinitis pigmentosa. Brain MRIs showed the presence of a “molar tooth sign,” which classifies this syndrome as classic JS with retinal involvement. Linkage analysis showed linkage to Xpter-Xp22.2 and a maximum LOD score of 2.06 for marker DXS8022. Mutation analysis revealed a frameshift mutation, p.K948NfsX8, in exon 21 of OFD1. In an isolated male with JS, a second frameshift mutation, p.E923KfsX3, in the same exon was identified. OFD1 has previously been associated with oral-facial-digital type 1 (OFD1) syndrome, a male-lethal X-linked dominant condition, and with X-linked recessive Simpson-Golabi-Behmel syndrome type 2 (SGBS2). In a yeast two-hybrid screen of a retinal cDNA library, we identified OFD1 as an interacting partner of the LCA5-encoded ciliary protein lebercilin. We show that X-linked recessive mutations in OFD1 reduce, but do not eliminate, the interaction with lebercilin, whereas X-linked dominant OFD1 mutations completely abolish binding to lebercilin. In addition, recessive mutations in OFD1 did not affect the pericentriolar localization of the recombinant protein in hTERT-RPE1 cells, whereas this localization was lost for dominant mutations. These findings offer a molecular explanation for the phenotypic spectrum observed for OFD1 mutations; this spectrum now includes OFD1 syndrome, SGBS2, and JS.     

Defining a New Candidate Gene for Amelogenesis Imperfecta: From Molecular Genetics to Biochemistry

Amelogenesis imperfecta is a group of genetic conditions that affect the structure and clinical appearance of tooth enamel. The types (hypoplastic, hypocalcified, and hypomature) are correlated with defects in different stages of the process of enamel synthesis. Autosomal dominant, recessive, and X-linked types have been previously described. These disorders are considered clinically and genetically heterogeneous in etiology, involving a variety of genes, such as AMELX, ENAM, DLX3, FAM83H, MMP-20, KLK4, and WDR72. The mutations identified within these causal genes explain less than half of all cases of amelogenesis imperfecta. Most of the candidate and causal genes currently identified encode proteins involved in enamel synthesis. We think it is necessary to refocus the search for candidate genes using biochemical processes. This review provides theoretical evidence that the human SLC4A4 gene (sodium bicarbonate cotransporter) may be a new candidate gene.     

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.     

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.     

Rett syndrome molecular diagnosis and implications in genetic counseling

Rett syndrome is a rare genetic X-linked dominant disorder. This syndrome is the most frequent cause of mental retardation in girls. In the classical form of the disease, the presenting signs and the course of development are characteristic. However clinical diagnosis can be very difficult when the expression is not in the classical form. Mutations in MeCP2 are responsible for 80% of cases. When MeCP2 mutation is found in an index case, genetic counseling is similar to that in other X-linked dominant genetic diseases. However, mutations in this gene can cause a spectrum of atypical forms. On the other hand, other genetic conditions like translocations, sex chromosome numerical anomalies, and mutations in other genes can complicate genetic counseling in this syndrome. We present the first case of molecular diagnosis of Rett syndrome in Iran and discuss the recent developments in its genetic counseling.