Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype.

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.

Cognitive manifestations of Huntington disease in relation to genetic structure and clinical onset.

OBJECTIVE: To examine the cognitive manifestations of Huntington disease (HD) with respect to age, clinical onset, progression, and genetic analyses. DESIGN: Case series of people with HD or at risk (AR) for HD. SETTING: Movement disorders and medical genetics clinics. PARTICIPANTS: Volunteer sample of 50 patients with HD and 127 AR adults. MEASURES: Neuropsychological evaluation was conducted with multiple measures of cognitive function (intelligence, memory, attention, executive, spatial, language), strength, manual speed/dexterity, somatosensory function, and mood. Quantitative molecular genetic analysis by means of polymerase chain reaction was conducted on 31 patients with HD and 86 AR subjects. RESULTS: In clinical HD, cognitive impairment correlated with number of years affected but not age at onset. The linear regression had a negative intercept, suggesting impaired cognitive function by the time of onset. In AR gene carriers, lower cognitive performance correlated with more trinucleotide repeats. In clinical HD, trinucleotide repeats interacted with disease chronicity such that more repeats were associated with worse performance over time; the overall effect of this was small compared with the effect of disease chronicity alone. Except for one AR subject, mood state was not associated with cognitive performance in either patients with HD or AR subjects. CONCLUSIONS: Cognitive decline appears to start before clinical onset of HD and is correlated with the number of trinucleotide repeats. Subsequent cognitive decline is primarily a function of number of years affected, although there is evidence that the presence of more trinucleotide repeats is associated with faster deterioration.

Creatine kinase in trout male germ cells: purification, gene expression, and localization in the testis.

High creatine kinase (CK) activity (16.5 +/- 7.6 IU/mg) is present in trout spermatozoa. In order to partly characterize the CK isozyme predominantly present in sperm and to study the expression of this protein in spermatogenesis, we purified to homogeneity a CK (s-CK) from trout sperm, by nitrogen cavitation followed by two chromatography steps (DEAE-Trisacryl and Blue Sepharose). Specific antisera to 5-CK were developed. A cDNA encoding for a CK named TCK1, and whose transcript shows enhanced testicular expression, was previously isolated from trout testis (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). A CK subunit expressed in vitro by this cDNA cross-reacts with anti-s-CK. A 21-amino-acid residue sequence near the N-terminus of s-CK is identical to the cDNA-derived sequence of TCK1, which is unlike any previously reported CK sequence. Using in situ hybridization, the TCK1 mRNA was detectable in primary and secondary spermatocytes and in early spermatids. Immunohistochemical staining of testis and various organs revealed that s-CK was confined to testis and, in this organ, to late spermatids and spermatozoa. In gill, some cells exhibited a positive signal, but another study rules out the presence of s-CK in this organ (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). These results demonstrate that s-CK/TCK1 is a germ cell-specific protein, the transcription of which starts in meiotic germ cells, while translation starts in late spermatids.

Simple and efficient subtractive hybridization screening.

A method of subtractive hybridization screening is presented that does not rely on the availability of large amounts of poly(A)+ RNA. The usefulness of this method is demonstrated by the isolation of cDNA clones that are unique or present at enhanced levels in cDNA libraries derived from a psoriatic skin cell line. A very high level of enrichment for rare cDNA sequences is possible as shown by the isolation of cDNA clones present at a frequency of less than one in one hundred thousand (0.001%). This method is particularly useful in experimental systems where the amount of RNA available is limited.

The predicted protein sequence of a fish testis-specific alpha-tubulin cDNA shows conservation of isotype-specific amino acid substitutions.

The alpha-tubulin cDNA clone, pTUB5 (1536 bp), contains the entire coding sequence for an alpha-tubulin of 450 amino acids (mol. wt. 50,000) and is the first tubulin sequence to be reported for a fish. The transcript encoded by pTUB5 showed testis-specific expression. Its gene appears to be present at a single copy number in the trout tubulin multigene family. Comparison of the TUB5 amino acid sequence to the murine testis-specific alpha-tubulin (M alpha 3/7) revealed that eight of ten murine isotype-specific amino acid substitutions were in common. Certain of these substitutions are also conserved in other testis-specific alpha-tubulins and in alpha-tubulins of certain protozoans with flagella. The conservation of specific amino acids at these positions suggests that they may have important structural and or functional roles in the microtubules of axonemes.

First expression of protamine message in trout testis.

In situ hybridizations were performed using a biotinylated riboprobe complementary to protamine messenger RNA in order to directly examine the various cell types in the trout testis for the presence of protamine message. Computer-aided optical density measurements were used to provide estimates of transcript abundance for cells identified by their DAPI-labeled nuclei. Optically detectable protamine hybridization occurred only in spermatid cells. These findings are in accord with results obtained in other species which report protamine mRNA only in the post-meiotic spermatid cell; but they are in conflict with a previous study employing solution hybridization which noted that protamine message first appears in the spermatocytes of rainbow trout.

A novel creatine kinase cDNA whose transcript shows enhanced testicular expression.

A cDNA (TCK1) encoding a creatine kinase (CK) subunit has been isolated from a cDNA library prepared from rainbow trout testis poly(A) + RNA. The predicted amino acid sequence for this CK subunit showed a novel amino-terminal coding region. Northern blot analyses demonstrated preferential expression of this CK subunit in testis relative to other CK containing organs. Southern blot analysis suggested a single copy number for this CK gene.

Isolation of dynein heavy chain cDNAs from trout testis which predict an extensive carboxyl-terminal alpha-helical coiled-coil domain.

A Mg2+-dependent ATPase activity has been purified from trout sperm axonemes which has properties characteristic of a dynein ATPase. A polyclonal antiserum prepared against the dynein heavy chains has been used to isolate dynein heavy chain (DYHC) cDNAs from a trout testis lambda gt11 cDNA expression library. beta-galactosidase fusion proteins produced in lambda gt11 by these trout cDNAs cross-reacted with a heterologous anti-sea urchin dynein antiserum. Northern blot analyses demonstrated that the RNA transcripts detected have sizes (7.5 - 12 kb) consistent with those expected for the dynein heavy chains. All the DYHC cDNAs encode portions of a highly unusual DNA coding sequence comprised of 21 bp direct repeats. The predicted open reading frame of this repeat is Ile/Leu-His-Val-Ile-Gln-Tyr-Ser and is characteristic of an extensive alpha-helical coiled-coil domain. The presence of an in-frame translation termination codon indicates that this domain is located at the carboxyl-terminus of the DYHC. Southern blot analyses demonstrated a low, if not single, copy number for this gene and conservation of this domain in other vertebrates. DYHC transcripts reach their highest level in testis, but are also abundant in brain tissue.

The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

Selective loss of a family of gene transcripts in a hereditary murine cataract.

The eye lens of the Fraser mouse contains a dominantly inherited cataract with reduced amounts of seven distinct but homologous gamma crystallins encoded by a family of gamma-crystallin genes. The results of experiments with cultured lenses, cell-free RNA translation, and Northern blot hybridization indicated a specific loss of the family of gamma-crystallin messenger RNA's in the Fraser mouse lens. Southern blot hybridization of genomic DNA's from normal and Fraser mice showed no differences in gamma-crystallin coding sequences.

Characterization of abnormal proteins in the soluble lens proteins of CatFraser mice.

The murine mutation CatFraser causes a cataract in both the mutant heterozygote (C/+) and mutant homozygote (C/C). Our previous electrophoretic studies detected, in the lenses of both mutant genotypes, several proteins which did not appear to be present in normal lenses. Here we show, using more sensitive methods, that traces of these proteins previously characterized as abnormal are in fact present in normal lenses. Furthermore, these proteins have a primary structure very similar to that of the alpha-crystallins. We conclude that the mutation accelerates the degradation of alpha-crystallin, which in the normal lens proceeds at a very slow rate.

Abnormalities of crystallins in the lens of the CatFraser mouse.

We have analysed, by column chromatography and two-dimensional electrophoresis, the soluble proteins present in the lenses of normal mice and of mice heterozygous (Cat/+) and homozygous (Cat/Cat) for the CatFraser mutation which causes a dominantly inherited cataract. In Cat/+ and Cat/Cat lenses, the gamma-crystallins comprise a smaller fraction, and the alpha-crystallins a greater fraction, of the total crystallins present than found in normal lenses. These changes in composition involve all the subunits of each crystallin class and show a dosage effect, the change being greater in Cat/Cat than Cat/+ lenses. In both Cat/+ and Cat/Cat lenses, the beta H-crystallin aggregate is lost and subunits are present which are not detectable in normal lenses.

Suspension cultures of adult rooster hepatocytes with a sustained vitellogenic response.

Primary suspension cultures of hepatocytes from adult roosters were prepared by collagenase perfusion. The cells remained viable for better than 12h as assessed by vital dye exclusion, electron microscopy and protein synthesis. In addition to the spectrum of proteins observed in control cultures, hepatocytes from animals pretreated with 17 beta-oestradiol synthesized vitellogenin, low density lipoprotein and several other unidentified peptides. Vg production, as determined by direct immunoprecipitation, was maintained at levels comparable to those observed in vivo with oestrogen treated roosters. Attempts to attain secondary induction of vitellogenesis in vitro, however, using cells from animals in which the primary response had subsided, were not successful. Nonetheless, the results show as a first step, that suspension cultures of liver cells from adult birds can be prepared which remain viable and morphologically differentiated for an extended period.