Linkage analysis of the mutation locus in the eye lens obsolescence (Elo) mouse

To map precisely the mutation locus of eye lens obsolescence (Elo) on mouse chromosome 1, subsequent linkage analysis was achieved using backcross mating between 129/SvSl-Elo (Elo/+) and 129/SvSl (+/+). Mouse genomic DNAs from 17 strains including the Elo mutant mouse were first digested with several restriction enzymes and analyzed by hybridization using gamma 2- and gamma 4-crystallin cDNAs as probes. Restriction endonuclease DraI showed distinct RFLP patterns in both cases. When gamma 2-crystallin cDNA was used as the probe, two strong bands were observed at 4.0 and 2.4 kb in the majority of strains, but the former fragment shifted to the 3.4-kb position in 129/SvSl-Elo (Elo/Elo) and CFO. The polymorphism between 4.0- and 3.4-kb fragments corresponded to the gamma 1-crystallin locus (Cryg-1), and that of the 2.4-kb one, to the gamma 2-crystallin locus (Cryg-2). Mouse DNAs were also analyzed by hybridization using gamma 4-crystallin cDNA (Cryg-4). In this case, 3.4- and 3.0-kb fragments were observed in Elo and wild-type mice, respectively. The backcross offsprings were analyzed with respect to Elo, Idh-1, Cryg-1, and Cryg-4 loci. Among 223 mice analyzed, recombination between Elo and Idh-1 loci was observed in three offsprings; and that between Cryg-1 or Cryg-4 and Idh-1 loci, in one offspring. No recombination occurred between Cryg-1 and Cryg-4 alleles.     

Characterization and purification of the 94-kDa glucose-regulated protein

Increased synthesis of so-called glucose-regulated proteins (grp) of 78 and 94 kDa occurs in mammalian cells exposed to a variety of agents, including 2-mercaptoethanol, tunicamycin, agents which perturb calcium homeostasis, and amino acid analogs. Herein we describe a number of properties of 94-kDa grp (grp 94) and present a method for its purification to homogeneity. The protein, within the endoplasmic reticulum (ER), is modified by the addition of high mannose-containing oligosaccharides. The predicted amino acid sequence of grp 94, as determined by others, has revealed the protein to contain a putative transmembrane domain near its amino terminus, but in addition, a potential endoplasmic reticulum retention sequence (KDEL) at its COOH terminus. Consequently, the question of whether grp 94 exists as a transmembrane or luminal protein of the ER remains controversial. Results using isolated microsomes subjected to either limited proteolysis or lactoperoxidase-mediated iodination were consistent with the idea that the grp is a transmembrane protein. On the other hand, using the method of sodium carbonate extraction, grp 94 exhibited properties of both a luminal and integral membrane protein. These results raise the question of whether there exist two different forms of grp 94 within the ER.     

Lamprey 48-kDa lens protein represents a novel class of crystallins

SDS-PAGE revealed a major Mr 48 000 polypeptide of pI around 8 in the water-soluble fraction of lamprey lenses. It occurs as a monomeric protein, and its amino acid composition and tryptic peptides show no resemblances to alpha-, beta-, gamma- or delta-crystallin. Immunoblotting with antiserum against the 48-kDa protein revealed an immunologically related polypeptide of similar Mr in reptiles, several birds and a fish, but showed no cross-reactivity with any other water-soluble lens component. The 48-kDa protein is not detected in many birds and fishes, and in the investigated mammals and amphibians.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

Defect of sperm assembly in a neurological mutant of the mouse, wobbler (WR)

In the wobbler (WR) mouse, a neuromuscular mutant characterized by a motoneuron degeneration and male infertility, the cellular basis of the defect in spermiogenesis was studied by light and electron microscopy as well as by lectin binding. Spermatozoa of the wobbler mutant had rounded heads, and their motility was reduced. In histological sections of WR testes, spermatogenesis appeared normal up to the stage of round spermatids, but the elongation and flattening of the nucleus during late spermiogenesis did not occur. Numbers of spermatid nuclei in WR testes were reduced to 70%-80% of controls. The acrosomal marker glycoprotein, peanut agglutinin receptor, was synthesized, but the acrosomal membrane did not attach to the nucleus. The disturbance in spermiogenesis of the wobbler mouse is not due to impaired descent of the testis, nor to a lack of testosterone, and is distinct from that observed in other mouse mutants (quaking, QK; Purkinje cell degeneration, PCD) with combined neurological and spermiogenesis defects.     

cDNA sequence analysis of CP94: rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins.

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.     

Characterization of a protein containing D-aspartic acid in aged mouse lens

A protein containing D-aspartic acid (D-Asp) was isolated from water insoluble (WI) fraction of naturally aged mice lens. The molecular weight of this protein was estimated to be about 10000 by gel permeation chromatography. High content of serine and glycine was noteworthy and the two amino acids occupy about 50 % of the total amino acids in the protein containing D-Asp.     

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.     

A truncated mutant of the extrinsic 23-kDa protein that absolutely requires the extrinsic 17-kDa protein for Ca2+ retention in photosystem II

One function of the extrinsic 23-kDa protein in photosystem II (OEC23) is to retain Ca(2+ )and Cl(-), two essential cofactors for photosynthetic oxygen evolution. A truncated mutant of OEC23 (OEC23 Delta19) revealed that 19 residues of the N-terminus of OEC23 were necessary for Ca(2+ )retention but not for its proper interaction with OEC17, the extrinsic 17-kDa protein in photosystem II. The lost ability of OEC23 Delta19 to reconstitute the oxygen-evolving activity was partially restored by OEC17 binding, suggesting the involvement of OEC17 in Ca(2+ )retention in photosystem II.     

ERp99, an abundant, conserved glycoprotein of the endoplasmic reticulum, is homologous to the 90-kDa heat shock protein (hsp90) and the 94-kDa glucose regulated protein (GRP94)

We have isolated an expressible full-length cDNA clone encoding murine ERp99, an abundant, conserved transmembrane glycoprotein of the endoplasmic reticulum membrane. ERp99 is synthesized as a 92,475-kDa precursor containing 802 amino acids. It possesses a signal peptide of 21 amino acids which is cleaved cotranslationally. Analysis of the amino acid sequence deduced from the nucleotide sequence of the cDNA clone led us to propose a model for the orientation of ERp99 in the endoplasmic reticulum membrane. In this model, ERp99 possesses one membrane-spanning, stop transfer segment in the N-terminal region. The protein chain passes through the membrane only once, and approximately 75% of the protein remains on the cytoplasmic side of the ER membrane. Comparison of the ERp99 sequence to the sequence of other proteins revealed that ERp99 has extensive homology with the 90-kDa heat shock protein of Saccharomyces cerevisiae (hsp90) and the 83-kDa heat shock protein of Drosophila melanogaster. In addition, the N terminus of mature ERp99 is identical to that of the 94-kDa glucose regulated protein (GRP94) of mammalian cells.     

Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2)

Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg²⁺ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn⁹¹-Ile-Val-Thr⁹⁴-Pro-Arg-Thr⁹⁷-Pro-Pro-Pro-Ser¹⁰¹. MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91–104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position –2 or –3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.     

Electrophoresis of mutant proteins in inherited diseases

The electrophoretic approaches for detection of mutant proteins in inherited diseases are briefly reviewed and discussed. Mutation of a protein, known to be associated with a specific inherited disease, is detected by immunoblotting, immunoprecipitation or enzyme staining, combined with various electrophoretic techniques. Some instrumental and technological devices for two-dimensional electrophoresis have been reported for the screening of mutant proteins in diseases of currently unknown etiology.     

Developmental regulation of a constitutively expressed mouse mRNA encoding a 72-kDa heat shock-like protein

Multiple heat shock cognate (hsc70) cDNA clones were isolated from the mouse embryonal carcinoma cell line F9. They all encode a single 72-kDa protein, which is constitutively expressed in all mouse cell lines and tissues tested, and which is only slightly induced by hyperthermia. hsc70 RNA is very abundant in F9 stem cells and brain, but very little is found in 14-day-old embryos. Upon differentiation of F9 stem cells induced by retinoic acid and cyclic AMP, expression of the hsc70 gene decreases only slightly, suggesting that hsc70 is highly expressed in early mouse development and is then down-regulated towards the end of embryogenesis. In adult tissues only the brain retains the high level of hsc70 gene expression found in F9 stem cells. We also show that expression of hsc70 protein and clathrin is uncoupled in F9 cells, indicating that the uncoating activity of coated vesicles may not be the only function of hsc70 protein.     

Role of a germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization.

Sulfolipid-immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077-1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm-egg binding assay with in vitro-capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm-zona pellucida interaction. In this study, using the in vitro sperm-egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the in vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti-SLIP1 IgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum IgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining.     

Regulation of mouse lens fiber cell development and differentiation by the Maf gene

Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.     

Genetics and ultrastructure of a cytoplasmically inherited yellow mutant in soybeans

A chimeric plant was observed in the F₂ generation of a cross between a male-sterile line and a plant introduction homozygous for a chromosome interchange in soybeans [Glycine max (L.) Merr.]. F₃ progeny of this plant included one chimera, 36 yellow plants and 16 green plants. The yellow plants, which progressively turn green, were viable and fertile in field, greenhouse and growth-chamber environments. Reciprocal cross-pollinations were made between these yellow plants and four known nuclear yellow mutant plants, between these yellow plants and sibling green plants and between these yellow plants and unrelated green plants. Segregation data from F₁ and F₂ generations indicated cytoplasmic inheritance of the newly discovered yellow phenotype. Pollinations in which reciprocal F₁ hybrid plants were used as male or female parents were made with unrelated green plants. Observations in F₁ and F₂ generations substantiated the hypothesis of cytoplasmic inheritance. No interactions have been observed between this mutant and the various nuclear backgrounds. This is the first report of a cytoplasmically inherited mutant affecting plant color in soybeans. Exchange grafts were made between cytoplasmic yellow plants and sibling green plants and between cytoplasmic yellow plants and unrelated green plants. The phenotype was controlled by the scion, indicating that graft-transmissible agents were not involved. When grown in darkness, cytoplasmic yellow plants and normal green plants accumulated the same amount of protochlorophyllide. Cytoplasmic yellow plants grown in dim light accumulated slightly less chlorophyll than did their green siblings. Electron photomicrographs showed that the prolamellar body (a structure associated with synthesis of protochlorophyllide) and chloroplast ultrastructure were normal in the cytoplasmic yellow mutant. These observations led to the hypothesis that the synchrony involved in deposition of nuclear and cytoplasmic gene products during organelle development is impaired in this cytoplasmic mutant.     

Complicated colobomatous microphthalmia in the microphthalmic (mi/mi) mouse

A study of the development of the eye in the cinnamon mouse, homozygous for the gene for microphthalmia (mi), has shown that the microphthalmia is due to failure of secondary vitreous formation associated with a coloboma. The retina is dystrophic but there is a residual population of large ganglion cells and the optic nerve also contains ganglion cells. All these ganglion cells have cytoplasm similar to the retinal ganglion cells in the normal controls. It is postulated that they communicate with axons in the optic nerve. In addition, the outer epithelial layer of the eye cup, which normally becomes pigmented, forms retinal tissue in the homozygous mouse and this is also true of the dorsal part of the eyestalk near the eye.     

The structural organization and protein composition of lens fiber junctions

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.