Developmental regulation for collagen II gene expression in transgenic mice

In order to evaluate the involvement of the type II collagen regulatory sequences in development, we have injected a construct containing a toxin gene under the control of the rat type II collagen promoter and enhancer. The construct, pDAS10-DTA, contained the diphtheria toxin A chain gene under the control of type II collagen sequences which had been used previously to target cartilagenous tissues in transgenics. Inspection of developing fetuses at various stages of gestation revealed a high number of aborted implants as well as abnormally developing fetuses. These abnormal fetuses were of small size, had shortened and underdeveloped limbs, cleft palates, and generally resembled a phenotype similar to chondrodystrophic mice. Histological comparisons of normal and abnormal fetuses indicated a reduced amount of extracellular matrix surrounding chondrocytes, and a disorganized appearance of the tissue. These results suggest that the expression of the toxin has occurred in chondrocytes and altered the survival and development of the transgenic mice. These results also indicate that the promoter and enhancer sequences contained in the transgene controlled the developmental expression of the type II collagen gene expression.     

Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii)

Seventeen desert tortoises, Xerobates agassizii, with upper respiratory tract disease were examined; thirteen were euthanatized for necropsy. Four normal control desert tortoises from a clinically healthy population were similarly evaluated. Hemoglobin and phosphorus values were significantly (P less than or equal to 0.05) lower and serum sodium, urea, SGOT, and cholesterol values were significantly higher in ill tortoises compared to controls. No significant differences in concentrations of serum or liver vitamins A and E were found between the two groups. While no significant differences were found for concentrations of lead, copper, cadmium, and selenium, the livers of ill tortoises had higher concentrations of mercury and iron. Lesions were found consistently in the upper respiratory tract (URT) of ill tortoises. In all ill tortoises dense infiltrates of lymphocytes and histiocytes obscured the mucosal epithelium and underlying glands. The mucosal epithelium was variably dysplastic, hyperplastic, and occasionally ulcerated. Electron microscopic studies revealed small (350 to 900 nm), pleomorphic organisms resembling Mycoplasma sp., in close association with the surface epithelium of the URT of ill tortoises. Pasteurella testudinis was cultured from the nasal cavity of all ill tortoises and one of four control tortoises. A Mycoplasma sp. was cultured from the nasal passageways of four ill tortoises and was ultrastructurally similar to the pleomorphic organism present on the mucosa in tissue section.     

Alternatively spliced RNAs encode several isoforms of CD46 (MCP), a regulator of complement activation

Five alternative cDNA clones were isolated for CD46, also known as the membrane cofactor protein (MCP) for the factor I-mediated cleavage of the complement convertases. One of these cDNA clones (a) was identical to an earlier MCP clone. The other four CD46 clones 3ontained the four NH₂-terminanl short consensus repeat (SCR) units of MCP, but differed at the region encoding the carboxyl-terminal of the protein which includes an extracellular segment rich in Ser, Thr, and Pro residues, a hydrophobic membrane-spanning domain, and a 33 amino acid cytoplasmic tail. The different CD46 cDNAs have variously: (b) inserted a 93 base pair (bp) exon resulting in a new cytoplasmic tail of 26 amino acids; (c) deleted a 42 bp exon from the extracellular Ser/Thr rich region; (d) used a cryptic splice acceptor sequence to delete 37 bp from an exon encoding transmembrane sequence; or (e) failed to splice the intron after the four SCR units. These were shown by northern blot and polymerase chain reaction to arise by alternative splicing of CD46 RNA. Forms (a), (b), and (c) of CD46 RNA are common in placental RNA, but (d) was rare, and (e) was incompletely processed and therefore aberrant. The polymerase chain reaction (PCR) was used to map the sites of the intron/exon junctions and demonstrate further possible splice variants of CD46. The alternative RNAs for CD46 may correlate to the different isoforms of CD46 found in different tissues, tumors, and in serum.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58050. Address correspondence and offprint requests to: D. F. J. Purcell.     

Microbial populations and hydrocarbon biodegradation potentials in fertilized shoreline sediments affected by the T/V Exxon Valdez oil spill.

The effort of clean up the T/V Exxon Valdez oil spill in Prince William Sound, Alaska, included the use of fertilizers to accelerate natural microbial degradation of stranded oil. A program to monitor various environmental parameters associated with this technique took place during the summer of 1990. Microbiological assays for numbers of heterotrophic and oil-degrading microbes and their hydrocarbon mineralization potentials were performed in support of this program. Fertilizer addition resulted in higher hexadecane and phenanthrene mineralization potentials on treated plots than on untreated reference plots. Microbial numbers in treated and reference surface sediments were not significantly different immediately after the first nutrient application in May 1990. However, subsurface sediments from treated plots had higher numbers of hydrocarbon degraders than did reference sediments shortly after treatment. The second application of fertilizer, later in summer, resulted in surface and subsurface increases in numbers of hydrocarbon degraders with respect to reference sediments at two of the three study sites. Elevated mineralization potentials, coupled with increased numbers of hydrocarbon degraders, indicated that natural hydrocarbon biodegradation was enhanced. However, these microbiological measurements alone are not sufficient to determine in situ rates of crude oil biodegradation.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.

     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Development of left/right handedness in the chick heart.

The chick heart tube develops from the fusion of the right and left areas of precardiac mesoderm and in almost all cases loops to the embryo's right-hand side. We have investigated whether any intrinsic difference exists in the right and left areas of precardiac mesoderm, that influences the direction of looping of the heart tube. Chick embryos incubated to stages 4,5 and 6 were cultured by the New method. Areas of precardiac mesoderm were exchanged between donor and host embryos of the same stage and different stages to form control, double-right and double-left sided embryos. Overall, double-right sided embryos formed many more left-hand loops than double-left sided embryos. At stages 4 and 5 a small percentage of double-right embryos formed left-hand loops (13%) whereas at stage 6 almost 50% of hearts had left-hand loops. Control embryos formed right-hand loops in 97% of cases. The stability of right-hand heart looping by double-left sided embryos, may be related to the process of 'conversion', whereas the direction of looping by double-right sided embryos has become randomised. There is some indication that an intrinsic change occurred in the precardiac mesoderm between stages 5 and 6 that later influenced the direction of looping of the heart tube. The direction of body turning is suggested to be linked to the direction of heart looping.     

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger.

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single KM(+)-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.