First record of soybean stem fly Melanagromyza sojae (Diptera: Agromyzidae) in Paraguay confirmed by molecular evidence.

We provided the first scientific record of Melanagromyza sojae (Zehntner, 1900), through molecular characterization of partial mtDNA COI gene, that confirms the occurrence of this pest in Paraguay. Previously reported in Brazil, an outbreak of larvae of M. sojae known as the soybean stem fly (SSF) that belongs to the family Agromyzidae, was also noted in soybean fields from the Canindeyú, Alto Paraná and Itapúa Departments in Paraguay. This pest is highly polyphagous, attacking various host plant species from the family Fabaceae, such as soybean and other beans. The implications of SSF detection in Paraguay are discussed in relation to the current soybean cultivation practices from this agriculturally important South American region, including Brazil.

Soybean Stem Fly, Melanagromyza sojae (Diptera: Agromyzidae), in the New World: detection of high genetic diversity from soybean fields in Brazil.

Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner), belongs to the family Agromyzidae and is highly polyphagous, attacking many plant species of the family Fabaceae, including soybean and other beans. SSF is regarded as one of the most important pests in soybean fields of Asia (e.g., China, India), North East Africa (e.g., Egypt), parts of Russia, and South East Asia. Despite reports of Agromyzidae flies infesting soybean fields in Rio Grande do Sul State (Brazil) in 1983 and 2009 and periodic interceptions of SSF since the 1940s by the USA quarantine authorities, SSF has not been officially reported to have successfully established in the North and South Americas. In South America, M. sojae was recently confirmed using morphology and its complete mitochondrial DNA (mtDNA) was characterized. In the present study, we surveyed the genetic diversity of M. sojae, collected directly from soybean host plants, using partial mtDNA cytochrome oxidase I (COI) gene, and provide evidence of multiple (>10) maternal lineages in SSF populations in South America, potentially representing multiple incursion events. However, a single incursion involving multiple-female founders could not be ruled out. We identified a haplotype that was common in the fields of two Brazilian states and the individuals collected from Australia in 2013. The implications of SSF incursions in southern Brazil are discussed in relation to the current soybean agricultural practices, highlighting an urgent need for better understanding of SSF population movements in the New World, which is necessary for developing effective management options for this significant soybean pest.

Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

Phylogeography Approach of Diloboderus abderus (Coleoptera: Melolonthidae) in the Southern Cone of America.

Diloboderus abderus (Sturm, 1826) (Coleoptera: Melolonthidae) is a serious soil pest of corn, wheat, oat, and natural and cultivated pastures in Argentina, Paraguay, Uruguay, and southern Brazil. Despite its economic importance, the genetic diversity and population structure of D. abderus remain unknown. We sequenced a fragment of the mitochondrial gene cytochrome oxidase I region (COI), of six populations of D. abderus from the Southern Cone of America. The mtDNA marker revealed a high haplotype diversity, high pairwise FST values, and significant genetic variations among populations. No correlation was found between genetic and geographical distances, yet the most common haplotype (Dab01) was present in four out of the six populations. Analysis of molecular variance showed that most of the variation was within populations of D. abderus. Tajima's D and Fu's FS tests indicated no evidence that D. abderus populations are under recent expansion. Our results indicate that genetic-based traits will likely remain localized or spread slowly, and management strategies need to be undertaken on a small scale.

Hormonal induction of transfected genes depends on DNA topology.

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Characterization of rearranged Y chromosomes in human testicular tumor cell lines.

Cytogenetic analysis of four cell lines established from two different human testicular tumors revealed rearranged or missing Y chromosomes. Southern blot analysis and in situ hybridization with different Y-derived human DNA sequences revealed the existence of Y chromosomal material even in a line without a cytogenetically visible Y chromosome and clarified the composition of Y marker chromosomes.

No association between the deltaF508 cystic fibrosis mutation and type 2 diabetes mellitus.

Cystic fibrosis (CF) is one of the most common recessively inherited disorders in Caucasian populations and is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. A three base deletion known as deltaF508 occurs on about 70%, of CF chromosomes and accounts for the high prevalence of the disease. Since type 2 diabetes mellitus occurs more frequently in relatives of patients with CF than in the normal population, we addressed the hypothesis whether heterozygosity for deltaF508 might be a genetic risk factor for type 2 diabetes. We screened 301 patients with type 2 diabetes mellitus which had been treated for at least three years from diagnosis by diet or oral antihyperglycemic agents. Healthy controls (n = 282) had no family history for diabetes. The genotype distribution did not differ significantly between patients with type 2 diabetes (2% heterozygotes) and controls (3% heterozygotes). According to these results, we conclude, that the deltaF508 mutation in its heterozygous form does not represent a major genetic risk factor for type 2 diabetes mellitus.

[Cystic fibrosis--initial diagnosis in a 39-year-old patient]. / Zystische Fibrose--Erstdiagnose bei einer 39-jährigen Patientin.

BACKGROUND: Cystic fibrosis is the most common hereditary disorder among Caucasians. Most of the patients are diagnosed as children. However, some cases are going undiagnosed into adulthood and are then often misdiagnosed because the non-pediatricians do not know cystic fibrosis very well and do not consider this diagnosis in adult patients. CASE REPORT: We present the medical history of a woman, who was diagnosed with cystic fibrosis at the age of 39 years, although she had suffered from bronchiectasis, pancreatic insufficiency and liver cirrhosis since many years. Her medical history was long with some diagnosis, but because of her age nobody considered the final diagnosis. CONCLUSION: In adult patients with bronchiectasis, liver cirrhosis and pancreatic insufficiency in combination or with only one of these symptoms, cystic fibrosis should be included into the differential diagnosis.

Synthesis and characterization of a DNA complementary to pre-uteroglobin mRNA.

Uteroglobin, a progesterone-induced uterine protein of the rabbit, is synthesized in cell-free systems as a precursor containing 21 additional amino-acids at its N-terminal end. The mRNA for pre-uteroglobin has been purified from the membrane-bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of r0t (0.01 M . s) and digestion of the non-hybridized cDNA by S1 nuclease. A comparison of the hybridization kinetics of the pre-uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre-uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamaide. The melting temperature of the hybrids of pre-uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA has been used to quantify pre-uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobin-synthesizing activity, there is a dramatic increase in the uterine content of pre-uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on a DNA basis there is a 50--100-fold increase in the cellular content of pre-uteroglobin mRNA following hormonal treatment.

Purification and properties of rabbit uterus preuteroglobin mRNA.

The mRNA for preuteroglobin, a precursor of the hormonally induced protein uteroglobin, has been partially purified from the endometrium of progesterone treated rabbits. The purification procedure starts with total endometrial polysomes and involves treatment with proteinase K and dodecylsulfate, chromatography on oligo(dT)-cellulose, sucrose density gradient centrifugation, electrophoresis in polyacrylamide gels containing dodecylsulfate, and a second absorption to oligo(dT)-cellulose. The final mRNA preparation codes exclusively for preuteroglobin in a wheat germ cell-free system and migrates as a single band in polyacrylamide gels containing 99% formanide. The average length of the poly(A) segment is 60 nucleotides and the translation of the preuteroglobin mRNA is inhibited by m7G(5')ppp(5')A, indicating that it contains a "capped" 5'-terminus. Comparison with known standards yields a molecular weight of 200,000 (600 nucleotides) for preuteroglobin mRNA, approximately twice as many nucleotides as required for encoding the 90 aminoacids of its cell-free product.

Cloning and sequence analysis of a human Y-chromosome-derived, testicular cDNA, TSPY.

The human Y-specific gene TSPY (testis-specific protein Y-encoded) was originally defined by the genomic probe pJA36B2 (DYS14), which detects a poly(A)+ RNA transcript in human testis tissue. Using this probe we have now isolated the cDNA sequence pJA923 from a human testis cDNA library. Southern blot hybridization experiments with both probes yielded identical male-specific banding patterns, but sequence analysis revealed an overall homology of only 92.3%. It appears that pJA36B2 (DYS14) is a pseudogene to pJA923 (TSPY), as only pJA923-specific transcripts were discovered in testis mRNA. PCR analysis of genomic DNA from patients with specific primers confirmed the simultaneous presence of at least two independent loci on the proximal short arm of the Y chromosome.

The human gene (DSG3) coding for the pemphigus vulgaris antigen is, like the genes coding for the other two known desmogleins, assigned to chromosome 18.

Pemphigus vulgaris (PV) is a potentially lethal skin disease in which epidermal blisters occur as the result of the loss of cell-cell adhesion caused by the action of autoantibodies against a keratinocyte cell surface glycoprotein, the PV antigen (PVA). This latter protein is a member of the desmoglein subfamily of the cadherin superfamily of cell-cell adhesion molecules, present in the desmosome type of intercellular junction. The other two known desmogleins are DGI, which is a target antigen in another autoantibody-mediated blistering disease of the epidermis, pemphigus foliaceous, and HDGC, which is expressed in the basal layer of the epidermis and in the simple epithelium of, for example, the colon. Genes coding for DGI (DSG1) and HDGC (DSG2) have previously been assigned to human chromosome 18. We now present evidence, using a polymerase chain reaction assay, that DSG3, the gene coding for PVA, is assigned to the same chromosome.

Tandem arrangement of the closely linked desmoglein genes on human chromosome 18.

The desmogleins, together with the desmocollins, both members of the cadherin superfamily, are the adhesive proteins of the desmosome type of cell junction, characteristically found in epithelial cells. Three different human desmoglein isoforms are encoded by separate genes (DSG1, DSG2, and DSG3) located on chromosome 18q12.1. DSG2 has been shown to be the most widely expressed in all desmosome-containing tissues, whereas DSG1 and DSG3 are expressed only in certain tissues, mostly stratified squamous epithelia. The desmoglein isoforms are expressed in a stratification-related manner in human epidermis, DSG1 being suprabasally expressed and DSG3 at a lower level, while DSG2 expression is weak and basal. Yeast artificial chromosome clones carrying all three known human desmoglein genes have now been isolated. The smallest clone containing all three DSG genes was 275 kb, and the three desmoglein genes were clustered within a region of less than 150 kb. From the types of clone obtained and from restriction enzyme analysis the order of the DSG genes and their orientation was deduced to be 5'-DSG1-DSG3-DSG2-3'. There thus appears to be some correspondence between the order of DSG genes and their expression within tissues, raising the intriguing possibility that the organization of the desmoglein gene cluster is required for properly regulated gene expression.

Cloning of the cDNA (DSC1) coding for human type 1 desmocollin and its assignment to chromosome 18.

Desmosomes are adhesive epithelial junctions that contain two distinct classes of cadherin-related glycoproteins (desmogleins and desmocollins), both of which occur as several different isoforms whose expression is related to epithelial differentiation. We have now isolated cDNA clones encoding a human desmocollin that is expressed in the more differentiated layers of human epidermis. This isoform has 53% amino acid identity with the previously isolated human (type 3) desmocollin, which is expressed in the basal layers of the epidermis. However, the N- and C-termini of the mature proteins are more highly conserved. Using a panel of somatic cell hybrids, human type 1 desmocollin (gene DSC1) has been assigned to chromosome 18, the same location as the other desmocollin gene (DSC3) and the three desmoglein (DSG) genes already mapped.

A sterile male with 45,X0 and a Y;22 translocation.

Cytogenetic analysis of a 20-year-old sterile male revealed a 45,X0 karyotype with no evidence for Y-chromosomal material on any of the chromosomes analysed by Q-, G- and C-banding. DNA analysis with 17 different Y chromosome-derived probes revealed the presence of Yp DNA sequences in the patient's genome. In situ hybridization with the Yp-derived probe pJA36B disclosed a translocation of Y-chromosomal material onto the short arm of a chromosome 22.

Chromosomal assignment of the human genes coding for the major proteins of the desmosome junction, desmoglein DGI (DSG), desmocollins DGII/III (DSC), desmoplakins DPI/II (DSP), and plakoglobin DPIII (JUP).

We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).