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Biomedical subjects

C L Holt

Publications and source records attributed to C L Holt.

8 recordsLinked to original sources

An extragenic suppressor of the mitosis-defective bimD6 mutation of Aspergillus nidulans codes for a chromosome scaffold protein.

We previously identified a gene, bimD, that functions in chromosome segregation and contains sequences suggesting that it may be a DNA-binding protein. Two conditionally lethal mutations in bimD arrest with aberrant mitotic spindles at restrictive temperature. These spindles have one-third the normal number of microtubules, and the chromosomes never attach to the remaining microtubules. For this reason, we hypothesized that BIMD functioned in chromosome segregation, possibly as a component of the kinetochore. To identify other components that function with bimD, we conducted a screen for extragenic suppressors of the bimD5 and bimD6 mutations. We have isolated seven cold-sensitive extragenic suppressors of bimD6 heat sensitivity that represent three or possibly four separate sud genes. We have cloned one of the suppressor genes by complementation of the cold-sensitive phenotype of the sudA3 mutation. SUDA belongs to the DA-box protein family. DA-box proteins have been shown to function in chromosome structure and segregation. Thus bimD and the sud genes cooperatively function in chromosome segregation in Aspergillus nidulans.

Alleles

A novel phage lambda replacement Cre-lox vector that has automatic subcloning capabilities.

We have developed a novel phage lambda replacement cloning vector, lambda pAn. lambda pAn allows one to automatically subclone the insert as a plasmid using the Cre-loxP site-specific recombination system. This eliminates the need to subclone insert fragments and permits the rapid structural analysis of insert DNA. lambda pAn is similar to other phage lambda replacement vectors taking inserts ranging in size from 5 to 19 kb. We have placed the pyrG gene of Aspergillus nidulans on the vector as a nutritional selective marker for transformation. We have developed this vector as part of an overall plan to facilitate the cloning of dominant extragenic suppressor mutations from A. nidulans, but also know that it is a generally useful vector for the purposes of isolating genomic clones without the need to subclone from the phage lambda vector.

Aspergillus nidulans

The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis.

A conditionally lethal mutation in the bimB gene of Aspergillus nidulans disrupts the normal regulatory patterns associated with mitotic events. This results in DNA replication in the absence of the completion of mitosis in the mutant at restrictive temperature. This defect yields large polyploid nuclei after several hours at restrictive temperature. The bimB gene has been cloned by genetic mapping and chromosome walking from the previously cloned amdS gene. The cloned DNA complements the temperature-sensitive recessive bimB3 mutation. Sequence analysis of overlapping complementary DNA clones for bimB predicts a polypeptide of 2,068 amino acids. The predicted polypeptide of 227,958 Da is shown to have a carboxyl-terminal region similar to those of the budding yeast ESP1 and fission yeast cut1+ genes. In contrast these genes exhibit no other regions of similarity to one another. The conserved domain in these three proteins and the similarity of the terminal mutant phenotypes for these genes are suggestive of a conserved function for this domain in each of the predicted polypeptides. We also present evidence for a second gene in the genome of A. nidulans which also has this conserved carboxyl-terminal region, suggesting that bimB, ESP1, and cut1+ may be members of a small gene family.

Amino Acid Sequence

The significance of portal vein gas in necrotizing enterocolitis.

To assess the significance of the presence of portal vein gas (PVG) in necrotizing enterocolitis (NEC), a retrospective study of all cases of NEC seen at Grady Memorial Hospital between July 1980 and June 1984 was conducted. Infants with PVG were compared with those without PVG with respect to clinical presentation, treatment, and outcome. There was no significant difference between the two groups in birth weight, Apgar scores, or the age of onset of NEC. All patients received standard medical therapy unless they developed pneumoperitoneum, had a positive paracentesis, or deteriorated in spite of maximal medical treatment, in which case they had surgery. Of 97 infants with "definite" NEC, 55 (56%) required surgery. Twenty-one patients (22% of the total) had PVG and 17 (81%) of them required surgery. Seventy-six patients (78% of total) did not have PVG and only 38 (50%) of them required surgery (P = 0.011). Five (29%) of 17 infants with PVG had nearly total intestinal necrosis compared with only five (13%, P = 0.25) of 38 infants without PVG. Patients with PVG had a lower overall survival rate (62% vs 88%, P = 0.009), medical survival rate (75% vs 97%, P = 0.184), and surgical survival rate (59% vs 79%, P = 0.19) than patients without PVG. Infants weighing less than 1000 grams fared worse regardless of the presence or absence of PVG. Portal vein gas is an ominous prognostic sign. It signals a greater likelihood for surgical intervention, a more extensive bowel involvement, and a significantly lower survival when compared to patients with NEC who do not have PVG.

Enterocolitis, Pseudomembranous

The role of host factors in an outbreak of necrotizing enterocolitis.

During an outbreak investigation of necrotizing enterocolitis (NEC) in a neonatal intensive care unit, we identified nine definite and six suspected cases of NEC on the basis of histopathologic, clinical, and roentgenographic findings. Neonates of low birth weight (less than 1,250 g) had the highest incidence of NEC, supporting a role for prematurity in this disease. Patients with definite NEC and those with severe clinical features had significantly lower birth weights and postconception ages (gestational age at birth plus postnatal age at onset of NEC) than the patients with suspected NEC. In a case-control study using birth weight-matched control subjects, maternal toxemia was identified as a possible protective factor for NEC. To our knowledge, this is the first report of the relationship between NEC disease severity and postconception age. These findings also suggest that toxemia may be an important protective factor in NEC and should be examined in subsequent studies.

Disease Outbreaks

The degradation of [14C]molinate in soil under flooded and nonflooded conditions.

The degradation of [14C]molinate in soil under flooded and nonflooded conditions was investigated. In laboratory studies, 50 percent of the applied molinate was lost in 3 weeks under moist soil conditions, while under flooded conditions, dissipation was reduced to 50 percent loss in 10 weeks. Analysis for molinate and its degradation products in the soil and flood water showed that under flooded conditions very little breakdown of molinate occurred, and volatilization was the primary mode of dissipation. However, under nonflooded conditions, hydroxylation of the azepine ring and further oxidation to the respective ketones occurs. Oxidation to form the sulfoxide, cleavage to yield the imine, and acetylation of the imine are also proposed. Other pathways appear to involve desethylation to produce a free thiocarbamic acid derivative followed by S-methylation as well as carboxylation of the S-ethyl group.

Azepines