Tubulin tyrosine ligase-like 1 deficiency results in chronic rhinosinusitis and abnormal development of spermatid flagella in mice.

Tubulin tyrosine ligase-like 1 (TTLL1) protein is a member of the tubulin tyrosine ligase superfamily of proteins that are involved in the posttranslational polyglutamylation of tubulin in axonemal microtubules within cilia and flagella. To investigate the physiological role of TTLL1, the authors generated mice with a gene trap mutation in the Ttll1 gene that provide confirmation in a mammalian model that polyglutamylation plays an important role in some ciliary and flagellar functions. For the first time, mice homozygous for the Ttll1 mutation exhibited accumulations of exudates in the nasal passages and sinuses, rhinosinusitis, otitis media, and male infertility. In homozygous mutant male mice, abnormal sperm morphology and function were characterized by shortened or absent flagella and immotility. Although homozygous mutant males were infertile, the females were fertile. These findings are consistent with a diagnosis of primary ciliary dyskinesia (PCD) resulting from ciliary dysfunction. They indicate that Ttll1 is essential for normal motility of respiratory cilia and the biogenesis and function of sperm flagella but that the defect does not result in the hydrocephalus or laterality defects often seen in other forms of PCD. The absence of early-onset lethal hydrocephalus in Ttll1-mutant mice may enable studies to evaluate the long-term effects of PCD in the respiratory system of mice. Although no mutations in the orthologous gene have been linked with PCD in humans, investigating the role of TTLL1 and polyglutamylation of tubulin in cilia and flagella should advance an understanding of the biogenesis and function of these organelles in mammals and have potential diagnostic and therapeutic applications.

Expression and purification of recombinant human zona pellucida proteins.

Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described.

The expression and localization of zona pellucida glycoproteins and mRNA in cynomolgus monkeys (Macaca fascicularis).

The zona pellucida (ZP) is an extracellular matrix composed of multiple glycoproteins that surrounds all mammalian oocytes. Analysis of the genes encoding the ZP proteins indicates that there are three classes ZPA (largest), ZPB (intermediate) and ZPC (smallest). Polyclonal antibodies developed against recombinant proteins of human and pig ZP have been used to identify immunohistochemically the components of the ZP of cynomolgus monkeys during the course of follicular development. Digoxigenin-labelled cDNA probes specific for the mRNA encoding ZPA, ZPB or ZPC were used to probe sections of cynomolgus monkey ovaries by in situ hybridization. mRNA encoding ZPA was identified in growing follicles at all stages and was seen in the granulosa cells of mature preovulatory follicles. ZPB protein and mRNA encoding ZPB were present in oocytes in secondary follicles and to a lesser extent in tertiary follicles, but were not found in primordial, primary or antral follicles or granulosa cells. ZPC protein and mRNA encoding ZPC were present in oocytes at all stages of folliculogenesis. These results suggest that each component of the ZP is produced and secreted at specific stages of folliculogenesis and that granulosa cells may be contributing to the synthesis of ZPA and ZPC.

Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families.

Full length zona pellucida cDNAs from cat, dog and pig that are homologous to the ZP2/rc75 genes from mouse, human and rabbit, a full length zona pellucida cDNA from cat and a gene and full length cDNA from human that are homologous to the rc55/ZP3 alpha genes from rabbit and pig, and full length zona pellucida cDNAs from cat, cow, dog, pig and rabbit that are homologous to the ZP3 genes from mouse, hamster, human and marmoset have been cloned and characterized. The members of these gene families are herein referred to as ZPA, ZPB and ZPC genes to avoid the confusion that currently exists in the zona pellucida of nomenclature. This report is the first to describe the presence all three major zona pellucida genes within individual mammalian species. Within the ZPA, ZPB and ZPC gene families, the DNA and deduced amino acid sequences are highly homologous to each other, and are most homologous between members of the same order within the class mammalia. These results imply that all or most mammalian species express the ZPA, ZPB and ZPC proteins, which form the zona pellucida layer surrounding the oocyte.

Nucleotide sequence of cDNA encoding ZP3 alpha, a sperm-binding glycoprotein from zona pellucida of pig oocyte.

We isolated a cDNA encoding the pig oocyte zona pellucida protein ZP3 alpha from a pig ovary lambda gt11 cDNA library. The 1699 bp cDNA contains a short 3' untranslated region characteristic of cDNAs encoding zona proteins. The deduced amino acid sequence for ZP3 alpha consists of 536 amino acid residues and shares 66% overall identity with a 55 kDa rabbit zona protein. Important features of the ZP3 alpha polypeptide include a predicted N-terminal signal sequence, twenty-two cysteine residues, an O-glycosylated domain and potential attachment sites for five N-linked sugar chains. A multibasic tetrapeptide occurs upstream of a predicted C-terminal transmembrane sequence; this suggests proteolytic processing of an integral membrane precursor within the constitutive secretory pathway.

PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains.

HIV-1 replication requires limited proteolysis of gag and gag-pol encoded precursor proteins by a specific viral proteinase (PR). Sequences of 20 different HIV-1 strains were compared in order to determine regions of conservation and variability within the PR gene. Viral strains included: (a) five new ones derived from New Orleans patient isolates, (b) four established ones grown in our laboratory, (c) eight, whose sequences were published in the Los Alamos Data Base (1990), (d) one Ugandan, and (e) two Brazilian isolates. In the first two groups, HIV proviral DNA extracted from infected lymphocytes was grown in tissue culture and directly amplified by PCR using specific primers flanking the PR gene. Amplified DNA was directly sequenced using a modified di-deoxy sequencing procedure. Sequence data showed a 25% variation among the 20 different HIV strains studied at the amino acid level, including 8% nonconservative changes and 17% conservative changes. Moreover, five noncontiguous regions were able to be delineated in which the PR showed no amino acid changes. These areas included amino acids (I) 1-9 (amino terminal sequence); (II) 21-32 (sequence around the active site); (III) 47-56 (top of the flap); (IV) 78-88; and (V) 94-99 (carboxy terminal sequence). Our results are consistent with those obtained from X-ray crystallography studies as well as single site mutational analysis.