Isolation and characterization of the human tyrosine aminotransferase gene.

Structure and sequence of the human gene for tyrosine aminotransferase (TAT) was determined by analysis of cDNA and genomic clones. The gene extends over 10.9 kbl and consists of 12 exons giving rise to a 2,754 nucleotide long mRNA (excluding the poly(A)tail). The human TAT gene is predicted to code for a 454 amino acid protein of molecular weight 50,399 dalton. The overall sequence identity within the coding region of the human and the previously characterized rat TAT genes is 87% at the nucleotide and 92% at the protein level. A minor human TAT mRNA results from the use of an alternative polyadenylation signal in the 3' exon which is present but not used at the corresponding position in the rat TAT gene. The non-coding region of the 3' exon contains a complete Alu element which is absent in the rat TAT gene but present in apes and old world monkeys. Two functional glucocorticoid response elements (GREs) reside 2.5 kb upstream of the rat TAT gene. The DNA sequence of the corresponding region of the human TAT gene shows the distal GRE mutated and the proximal GRE replaced by Alu elements.     

Purification and Characterization of an Active Form of the p78Protein of Adeno-associated Virus Type 2 Expressed inEscherichia coli

The 78-kDa product (p78^Rep) of therepgene of AAV-2 was expressed with an amino-terminal histidine-tag inEscherichia coliand was purified under denaturing conditions. After renaturation of the p78^Repprotein by serial steps of dialysis, the biochemical activitites of the p78^Repprotein were demonstrated, which include the ATP-dependent endonuclease and helicase activity as well as sequence-specific binding to the AAV-2 terminal repeat. These activities were retained when the protein was purified under denaturing conditions followed by renaturation. When compared with published data for p68^Rep, the helicase activity of p78^Repwas stronger and the endonuclease acitvtiy was weaker. The p78^Repprotein was able to inhibit HIV-1 replication after co-microinjection together with infectious proviral HIV-1 DNA into the nuclei of human cells, suggesting that p78^Repis necessary for inhibition of HIV-1 in vivo.     

Deletions and Point Mutations of LRRC50 Cause Primary Ciliary Dyskinesia Due to Dynein Arm Defects

Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.     

Remodelling of VipA/VipB tubules by ClpV‐mediated threading is crucial for type VI protein secretion

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae. T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N‐terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel‐like structures that are converted by a threading activity of ClpV into small complexes. ClpV‐mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion.     

Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis

Cilia formation is a multi-step process that starts with the docking of a vesicle at the distal part of the mother centriole. This step marks the conversion of the mother centriole into the basal body, from which axonemal microtubules extend to form the ciliary compartment. How vesicles are stably attached to the mother centriole to initiate ciliary membrane biogenesis is unknown. Here, we investigate the molecular role of the mother centriolar component Cep164 in ciliogenesis. We show that Cep164 was indispensable for the docking of vesicles at the mother centriole. Using biochemical and functional assays, we identified the components of the vesicular transport machinery, the GEF Rabin8 and the GTPase Rab8, as interacting partners of Cep164. We propose that Cep164 is targeted to the apical domain of the mother centriole to provide the molecular link between the mother centriole and the membrane biogenesis machinery that initiates cilia formation.