Hintergrund Der Begriff Primäre Ciliäre Dyskinesie (PCD) umfasst eine Gruppe seltener, genetisch bedingter Erkrankungen, die durch eine Dysfunktion motiler Zilien charakterisiert ist. Sie ist klinisch und genetisch heterogen, aktuell sind mehr als 50 Gene bekannt, die mit einer PCD assoziiert sind. Typischerweise kommt es durch eine gestörte mukoziliäre Reinigung zu einer Sekretretention und zu rezidivierenden Infektionen der oberen und unteren Atemwege, die eine dauerhafte Lungenschädigung zur Folge haben.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

In order to identify the underlying genetic defect in a cohort of individuals with chronic destructive airway disease and congenital brain malformation, we performed whole exome sequencing. We identified homozygous loss of function variants in seven individuals from five non-related families in TP73. TP73 encodes for the tumor protein 73. As member of the TP53 family of transcription factors, it has been well studied in cancer research. All patients harboring mutations in TP73 were suffering from a chornic airway disease and lissencephaly. Since analysis in mice had shown a multiciliogenesis defect in TP73 deficient mice, we next asked whether the respiratory phenotype was caused by a mucociliary clearance disorder. To this end we used a ciliary beat assay using an air liquid interface culture of respiratory epithelia obtained from affected individuals and healthy controls. This analyses revealed that cilia were not able to generate a sufficient fluid flow, conistent with impaired mucociliary clearance. Transmission electron microscopy and immunofluorescence analysis of respiratory epithelia before and after cell culture showed reduced ciliary length and basal bodies mislocalized in the cytoplasm. Consitent with a defect in cell differentiation/proliferation a reduced cell layer and a reduced number of ciliated cells were identified. Our data show that autosomal recessive mutations in TP73 cause a motile ciliopathy due to a defect in multiciliated cell differentiation. References: Wallmeier J, Bracht D, Alsaif HS, Dougherty GW, Olbrich H, Cindric S, Dzietko M, Heyer C, Teig N, Thiels C, et al. Mutations in TP73 cause impaired mucociliary clearance and lissencephaly. Am J Hum Genet 2021;1–12

Technological advances in molecular biology should make possible the resolution of the important structural features which control viral gene expression and hence viral phenotype. This chapter reviews gene expression of RNA plant viruses using established viral classification groupings. It discusses each virus group by briefly describing the structural organization of the virion and of the viral genomic RNA(s). The chapter discusses the nine virus groups that have a single-stranded, ribonucleic acid component which is infectious. The initial five virus groups have members possessing a particle morphology which is spherical. The remaining four virus groups have members which are anisometric in morphology. The chapter presents the expression of the viral RNA in cell-free systems and in the host cells. It integrates in vivo and in vitro observations and overviews the genomic expression of each virus group.

We present a stratification of the genetic basis of primary ciliary dyskinesia (PCD), based on screening >230 individuals for gene mutations using various approaches including whole exome sequencing. PCD is a genetically heterogeneous recessive ciliopathy, characterized by chronic lung disease and laterality and fertility defects arising from cilia and sperm dysmotility. Most PCD is caused by loss of the ciliary outer dynein arm motors (ODA) essential for motility, arising from mutations in ODA subunits or ODA docking and targeting proteins. Gene panel resequencing of candidate ciliopathy genes in affected children from a consanguineous Bedouin-Arabic family has recently revealed a homozygous protein truncating variant in CCDC151 (c.925G>T; p.Glu308*). Parallel exome sequencing combined with autozygosity mapping in a consanguineous UK-Pakistani-origin family highlighted a large autozygous region on chr 19p13 harbouring a homozygous CCDC151 protein-truncating variant (c.1256C>T; pSer419*). Sanger sequencing of CCDC151 in 150 more PCD cases identified another individual carrying c.925G>T. Transmission electron microscopy of respiratory cilia from individuals carrying CCDC151 mutations showed loss of ODA. Consistent with laterality defects in these individuals, we find Ccdc151 expressed in vertebrate left-right organizers. Both homozygous zebrafish and mouse Ccdc151-deficient mutants display situs defects associated with complex heart defects. Immunofluorescence analysis in patients shows that CCDC151 mutations abolish assembly of CCDC151 into respiratory cilia, and furthermore cause a failure in assembly of the ODA component DNAH5 and ODA docking proteins CCDC114 and ARMC4. We conclude that CCDC151 mutations appear to cause PCD by disruption of the axonemal ODA docking complex machinery.

The SEQALIGN programs de scr ibed in t h i s report aid in t h e a s s e m b l y of up t o 100 i n d i v i d u a l o v e r l a p p i n g DNA s e q u e n o e s generated by H-13 aubcloning and sequencing methods. The program produces a p r i n t o u t of the a l i gned sequences presented in r e g i s t e r . Use of t h e program w i l l be f a c i l i t a t e d b e c a u s e 1) i t i s written with the Microsoft BASIC interpreter, 2) sequence data may be entered and edited using WORDSTAR or similar word processing programs, and 3) hardware requirements for execution of the program on CP/H or MS-DOS (IBM-PC compatible) systems are minimal . INTRODUCTION. Various subcloning strategies (1,2) for determination of DNA sequence have increased the ease and rapidity with which sequence information can be generated. In these procedures, a large DNA clone is digested with restriction endonucleases, fragments are subcloned into an appropriate vector, and the fragments are sequenced individually. Using nucleases with different s p e c i f i c i t i e s , a series of overlapping fragments of the original DNA sequence is obtained. However, the location of each fragment within the complete sequence and the orientation of the fragment usually are not readily apparent. As manual assembly of these fragments into the complete sequence i s a time consuming and tedious undertaking, several computer programs have been devised which automatically or semi-automatioally assemble the fragments into a proposed consensus sequence (3-7). Such programs require access to mainframe or minicomputers. Other programs are offered as sections of expensive commercial software packages. It was our goal to write a ser ies of programs which SEQALIGN is available for a $50 contribution to the Microbiology Enrichment Fund, Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695-7615. © IRL Press Limited, Oxford, England. 5 1 7 Nucleic Acids Research would address the assembly problem yet bo accessible to virtually anyone using these sequencing techniques. This cons tra int re quired that the programs run on small CP/H or MS-DOS based microcomputers, that they be wri t ten in BASIC, and that they be capable of handling a s u f f i c i e n t number of fragments to be of s i g -

Treatment of tall and dwarf (3 beta-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19. In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line. Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feed-back regulation of the 20-oxidation and 3 beta-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product. The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression.

In many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. Proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. Recombinant DNA methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. Various systems and strategies have been successfully employed, depending on the specific protein of interest and the desired use of the final end product (antibody production, crystallography studies etc.). This chapter reviews some common methods for the production of recombinant fusion proteins and specifically describes a versatrle method for the removal of affinity tags from recombinant fusions using a highly purified proteinase with an unparalleled degree of specificity. This proteinase, from the genome of tobacco etch virus (TEV), demonstrates specific proteolytic activity under a wide range of parameters (salt, temperature, pH), making it an excellent choice for cleavage of fusion proteins (1,2).

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, were identified as pathotypes P-1 and P-4, respectively, by their infectivity on Pisum sativum L. lines homozygous for the recessive resistance genes sbm-1 and sbm-4. The two isolates differed in several biological characteristics. DPD1 induced transient vein clearing, downward rolling of leaflets and internode shortening on P. sativum, whereas NY only caused a slight growth reduction. DPD1 moved systemically in Chenopodium quinoa whereas NY was restricted to inoculated leaves. DPD1 was frequently transmitted by seeds whereas NY was rarely seed-transmitted: 24% and 0.3%, respectively, in P. sativum '549'. Both DPD1 and NY were transmitted by aphids (Myzus persicae), though a DAG triplet was not present in the N terminus of the coat protein. The nucleotide sequence and deduced amino acid sequence of NY were determined and compared to the corresponding sequences of DPD1.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.

Two pea seedborne mosaic potyvirus (PSbMV) isolates, P-1 DPD1 (P-1), which is highly seed-transmitted, and P-4 NY (P-4), which is rarely seed-transmitted, and chimeras between P-1 and P-4 were analysed to map the viral genetic determinants of seed transmission. Infectivity of chimeric viruses was evaluated by inoculating Pisum sativum with RNA transcribed in vitro from recombinant full-length cDNA clones. The chimeric viruses that were used demonstrated that a genomic segment encoding the 49 kDa protease and putative RNA polymerase was responsible for symptom induction. Attempts to determine transmission of the chimeric viruses in P. sativum cultivars known to transmit P1 at high frequencies showed that seed transmission is a quantitative character influenced by multiple viral determinants. Seed transmission frequency did not correlate with accumulation of virus in vegetative tissue. The 5' 2.5 kb of the 10 kb PSbMV genome had a major influence on the seed transmission frequency and was analysed further. This showed that, while the helper-component protease was a major determinant of seed transmission, the potyviral P1 -protease exerted no measurable influence.

Sense RNA-mediated virus resistance has been described for transgenic plants expressing potyviral capsid protein sequences. This study was undertaken to determine if expression of other viral sequences could induce this type of virus resistance. Plants showing highly resistant or 'recovery' phenotypes were generated by expressing the tobacco etch virus (TEV) 6 kDa/21 kDa reading frames. Expression of translatable or untranslatable versions of this TEV sequence produced resistant lines. Highly resistant and recovery phenotype plants expressing TEV coat protein sequences. High transcription rates with low steady-state levels of the transgene transcript generally correlated with resistance. During recovery and induction of the resistance, RNA and protein steady-state levels decreased 5- to 20-fold, while transcription of the transgene continued at a similar level. A posttranscriptional, cellular system eliminating sequences contained in the transgene transcript and viral genome would be consistent with the results.

Transgenes provide unique opportunities to assess the relationship between genotype and phenotype in an organism. In most cases, introduction and subsequent expression of a transgene will increase (with a sense RNA) or decrease (with an antisense RNA) the steady-state level of a specific gene product. However, a number of surprising observations have been made in the course of many transgenic studies. We develop a hypothesis that suggests that many examples of endogenous gene suppression by either antisense or sense transcripts are mediated by the same cellular mechanism.

The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not This conversion was dilution independent and thought to be intramolecular. Isolation of the ∼2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxyterminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.