Many random sequences functionally replace the secretion signal sequence of yeast invertase.

In the process of protein secretion, amino-terminal signal sequences are key recognition elements; however, the relation between the primary sequence of an amino-terminal peptide and its ability to function as an export signal remains obscure. The limits of variation permitted for functional signal sequences were determined by replacement of the normal signal sequence of Saccharomyces cerevisiae invertase with essentially random peptide sequences. Since about one-fifth of these sequences can function as an export signal the specificity with which signal sequences are recognized must be very low.

Post-transcriptional control of the Arabidopsis auxin efflux carrier EIR1 requires AXR1.

The auxin efflux carrier EIR1 (also known as AGR and AtPIN2) is a key mediator of the response of Arabidopsis roots to gravity [1,2]. This response is thought to require the establishment of a transient auxin gradient in the root meristem, resulting in differential cell elongation [3]. Recent reports suggest that EIR1 is essential for the asymmetric distribution of auxin in the root meristem [4-7], but the regulatory aspects of this process are still not fully understood. Here, we studied the regulation of EIR1 in Arabidopsis using two reporters: one was a translational fusion that contained the entire EIR1 coding sequence, and the other a transcriptional fusion that had no EIR1 coding sequence. We found that EIR1 is controlled at the post-transcriptional level. The translational fusion was unstable in response to changes in auxin homeostasis, and was destabilized by cycloheximide. In contrast, the protein was stabilized in the axr1-3 mutant, which is auxin resistant and defective in auxin responses such as root gravitropism [8,9]. AXR1 is thought to participate in ubiquitin-mediated control of protein stability [10-12]. The dependence of EIR1 reporter expression on auxin concentrations and AXR1 suggests that auxin transport is regulated through a feedback regulatory loop that affects protein stability in response to auxin.

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

Structure and function of the yeast URA3 gene: expression in Escherichia coli.

The expression of the cloned Saccharomyces cerevisiae URA3 gene in Escherichia coli on both plasmid and phage vectors was studied. Isolates of the gene from two different laboratory strains of yeast differ in their ability to be expressed in E. coli in the absence of external adjacent promoters of transcription. The DNA sequence of the two genes was determined and revealed several differences in the DNA flanking the structural gene. One base change alters the "Pribnow-box" of an E. coli promoter present in the yeast sequences. Three amber alleles of the yeast gene were also cloned from yeast. Two of the alleles could be suppressed in E. coli by a tRNA suppressor mutation. One of the amber alleles was determined to be a mutation in the seventh codon of the structural gene, thereby establishing the reading frame and extent of the coding sequence. The initiator codon of the reading frame encoding the URA3 structural gene is preceded by two other ATG codons in a different reading frame 61 and 79 bp away. The nearer ATG begins an open reading frame that overlaps the structural gene sequences by 17 bp. With the DNA sequence of the URA3 gene many of the common yeast vector plasmids are now completely known at the level of DNA sequence.

Bilateral bipartite medial cuneiform. A case report.

A fracture to the intermediate cuneiform that was not definitively detected on routine radiographs because of the overlap of the cuneiform was presented. Weightbearing x-rays did not provide additional information. The anatomical location of the fracture was identified only after a computed tomography scan was performed. An incidental finding, bilateral bipartite medial cuneiforms, was also observed on the computed tomography scan, which contributed to the overlap on routine radiographs. The anatomy of the bipartite medial cuneiforms seen on computed tomography was similar to that described by Barlow in 1942. Retrospective comparison to the initial radiographs with the computed tomography scan sections did reveal bipartite medial cuneiforms on these films as well. The podiatric physician should keep bipartition in mind when evaluating x-rays for any osseous pathology, especially fractures.

Pathophysiology of diabetic neuropathy.

Angiopathy, immunopathy, and neuropathy are the key components responsible for diabetic foot complications. The authors report on the current theories of metabolic and structural causes of diabetic neuropathy.

Pyoderma gangrenosum involving the foot. A case report.

Pyoderma gangrenosum is a rare and destructive inflammatory skin disease. The authors present a report of a patient with a classic case of pyoderma gangrenosum involving the foot. The diagnosis was made on the basis of clinical presentation and progression of the disease after differential diagnoses of common conditions were excluded. A brief overview of the disease process, its treatment, and its correlation with ulcerative colitis is provided.

Three select subungual pathologies: subungual exostosis, subungual osteochondroma, and subungual hematoma.

Special attention has been given to the differences between subungual exostosis and subungual osteochondroma. Once a diagnosis has been made and symptoms persist, complete excision of the lesion with curettage of the base is the treatment of choice for both conditions. Subungual hematoma is an exquisitely painful condition that usually results from trauma, but may also be caused by systemic pathology, medication and drug reactions, and aging. Prompt decompression will significantly reduce pain and further damage to the nail bed and matrix. Radiographs must be taken because of the high incidence of associated distal phalangeal fracture. Patients must be warned that onycholysis, transient and permanent nail deformity, and infection are possible complications, even with the best treatment.

Lower extremity manifestations of cardiovascular disease. An update.

Cardiovascular disease is one of the leading causes of morbidity and mortality, and its system-wide complications are numerous and complex. Many signs and symptoms of cardiovascular pathology are expressed in the lower extremities. This article addresses new research that relates the lower extremity with cardiovascular disease.

FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation.

FUS3 is required for both the arrest of cells in G1 and mating. Upon exposure to mating pheromone, fus3-1 and fus3-2 mutants fail to arrest in G1 and continue to divide while undergoing the transcription induction and morphological changes typical of mating cells. The G1 arrest defect of these fus3 mutants is suppressed by a daf1/whi1 null mutation (also called cln3, a putative cyclin). FUS3 has a positive role in conjugation, because overexpression of FUS3 increases the pheromone sensitivity of wild-type cells, while the absence of FUS3 causes sterility. The suppression of a gpa1 null (G alpha subunit) by a fus3 null also suggests FUS3 is in the signal transduction pathway. The predicted FUS3 protein is 35% identical to the cdc2+/CDC28 kinases and 52% identical to the KSS1 predicted kinase.

A pathway for lateral root formation in Arabidopsis thaliana.

In plants, the hormone indole-3-acetic acid (IAA) can initiate the developmental program for lateral root formation. We have isolated mutants that have permitted the dissection of this program into initiation and maturation of lateral roots. The alf1-1 mutation causes hyperproliferation of lateral roots, alf4-1 prevents initiation of lateral roots, and alf3-1 is defective in the maturation of lateral roots. The alf3-1 mutant can be rescued by IAA, whereas the alf4-1 mutant is not rescued. Our data suggest a model in which IAA is required for at least two steps in lateral root development: (1) to initiate cell division in the pericycle, and (2) to promote cell division and maintain cell viability in the developing lateral root.

Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.

The ends of Tn10 are not IS3.

By heteroduplex and hybridization analysis we showed that the inverted repetition (here called IS10) at the ends of the translocatable tetracycline resistance element Tn10 is not IS3, as had previously been reported by Ptashne and Cohen (J. Bacteriol. 122:776--781, 1975). Further analysis confirmed the homology between IS3 and the alpha beta sequence of F and demonstrated that IS10 was not present in the genomes of Salmonella typhimurium LT2 or Escherichia coli K-12.

Prevention and care of diabetic foot ulcers.

The foot is frequently overlooked in the management of diabetic patients. Failure to control diabetic foot ulcers at an early stage can lead to life-threatening infection or amputation. Preventive care should emphasize patient education, glycemic control, careful daily foot hygiene and appropriate footwear. Early management of a diabetic foot ulcer should include culture-directed antibiotic therapy when there is evidence of infection, moist dressings and adjustment of footwear or casting to avoid pressure on the wound site. All patients with foot ulcers should be evaluated for evidence of foot ischemia. Surgical intervention to debride infected tissue and bone or to revascularize ischemic tissue can aid in ulcer healing. Serious infection or severe ischemia, unfortunately, often necessitates amputation.

Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro.

Short single-stranded gaps can be constructed by limited exonuclease action at single-stranded breaks (nicks) placed at predetermined sites on closed circular DNA molecules. As efficient primer-templates for DNA polymerase, single-stranded gaps can be repaired in vitro to regenerate an intact DNA duplex. In this report two in vitro reaction schemes are described that produce a high frequency of errors during repair ("misrepair") of gaps and thereby allow the efficient recovery of mutations limited to the nucleotide sequence at or near the original gap. In the first of these misrepair schemes, nucleotide misincorporations are stimulated by omission of one of the four deoxynucleoside triphosphates; the misincorporations are trapped by the presence of excess DNA ligase in the reaction mixture. The second misrepair scheme involves the misincorporation of an excision-resistant alpha-thiophosphate nucleotide, followed by gap filling in the presence of all four conventional deoxynucleoside triphosphates. When applied to short gaps constructed at one of several unique restriction sites on the small plasmid pBR322, both gap misrepair methods yielded mutations within the targeted restriction site at high frequency (6--42%). A majority of the sequence changes identified were base substitutions; transversions and transitions are approximately equally represented. The remaining sequence changes were an insertion of a single base pair and deletions of one to four base pairs.

Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo.

A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken beta-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast beta-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the beta-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single beta-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain beta-tubulins.

Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization.

The alanine racemase encoded by the Salmonella typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37 degrees C the enzyme has a specific activity of 1400 units/mg (V max, L- to D-alanine). Active enzyme molecules are monomers of Mr 39 000 with one molecule of pyridoxal 5'-phosphate bound per subunit. The Km's for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated molecular weight (apoenzyme) was 39 044.

Yeast suppressors of UAA and UAG nonsense codons work efficiently in vitro via tRNA.

A cell-free protein-synthesizing system, containing an S-100 fraction from yeast, ribosomal subunits from Krebs ascites cells, and ribosome initiation factors from rabbit reticulocytes, translates yeast, adenovirus, and rabbit globin messenger RNAs and the RNA from bacteriophage Qbeta. An amber mutation in the Qbeta synthetase gene is suppressed in vitro if the S-100 fraction s from yeast strains carrying amber suppressor mutations. Suppressor SUP6-2 gives 16% suppression, and the recessive lethal suppressor RL-1 gives 50% suppression. Extracts from strain FM6, which has the ochre suppressor SUP4-1, give a longer protein product from the normal synthetase gene at Qbeta with an efficiency of 63%. This implies that UAA is the terminator for the synthetase gene, and that synthesis of this read through protein can be used as an assay for ochre suppression. Suppression in each of these cases is mediated by tRNA, since pufified tRNA is the only fraction from suppressing strains that is required in an otherwise nonsuppressing cell-free system.

Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes.

Wild-type Arabidopsis plants maintain a relatively constant size over a wide range of temperatures. Here we show that this homeostasis requires the BONZAI1 (BON1) gene because bon1 null mutants make miniature fertile plants at 22 degrees C but have wild-type appearance at 28 degrees C. The expression of BON1 and a BON1-associated protein (BAP1) is modulated by temperature. Thus BON1 and BAP1 may have a direct role in regulating cell expansion and cell division at lower temperatures. BON1 contains a Ca(2+)-dependent phospholipid-binding domain and is associated with the plasma membrane. It belongs to the copine gene family, which is conserved from protozoa to humans. Our data suggest that this gene family may function in the pathway of membrane trafficking in response to external conditions.

EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana.

The EIR1 gene of Arabidopsis is a member of a family of plant genes with similarities to bacterial membrane transporters. This gene is expressed only in the root, which is consistent with the phenotypes of the eir1 mutants-the roots are agravitropic and have a reduced sensitivity to ethylene. The roots of eir1 mutants are also insensitive to the excess auxin produced by alf1-1 and fail to induce an auxin-inducible gene in the expansion zone. Although they fail to respond to internally generated auxin, they respond normally to externally applied auxin. Expression of the EIR1 gene in Saccharomyces cerevisiae confers resistance to fluorinated indolic compounds. Taken together, these data suggest that the EIR1 protein has a root-specific role in the transport of auxin.