Characterization of the gene for ADP-ribosylation factor (ARF) 2, a developmentally regulated, selectively expressed member of the ARF family of approximately 20-kDa guanine nucleotide-binding proteins.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activities of cholera toxin in vitro and function in protein trafficking in vivo. The six cloned mammalian ARFs can be grouped into three classes based on size and sequence identity. ARF 2 is a class I ARF, whose approximately 2.6-kilobase mRNA exhibits species and tissue selective expression and is developmentally regulated in rat brain. Here we report the sequence, structure, and functional promoter region of the bovine ARF 2 gene, which was facilitated by constructing a composite cDNA. The ARF 2 cDNA, constructed from a partial cDNA clone and polymerase chain reaction-amplified fragments from reverse-transcribed poly(A)+ RNA, was approximately 2270 base pairs (bp) (minus the poly(A) tail). In the 3'-untranslated region, there are two potential polyadenylation signals, ATTAAA and AATAAA, at positions 1064 and 2232, respectively, and two ATTTA motifs, believed to signal mRNA degradation, at positions 2115 and 2165. The ARF 2 gene, represented in three overlapping genomic clones, spans approximately 20 kilobase pairs with five exons and four introns. Consensus sequences for guanine nucleotide-binding and GTP hydrolysis are in separate exons, except for the NKXD sequence, which is divided by intron 4. There are multiple transcriptional initiation sites. Transient transfection of embryonic trachea cells with deletion constructs defined the functional promoter region to be within 400 bp upstream of the most 5' site of transcription initiation. This 400-bp region lacks a TATA-like sequence but contains six inverted CCAAT boxes, four potential Sp1-binding sites, and a potential AP-2-binding site. Although the pattern of expression of ARF 2 is unique among the ARFs, the structures of the class I ARF genes are conserved among its members and across species.

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions.

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.

Expression of Go alpha mRNA and protein in bovine tissues.

Go alpha is a 39-kDa guanine nucleotide-binding protein (G protein) similar in structure and function to Gs alpha and Gi alpha of the adenylate cyclase complex and to transducin (Gt alpha) of the retinal photon receptor system. Although expression of Go alpha protein has been reported to be tissue-specific, other workers have found Go alpha mRNA in all rat tissues examined. In order to clarify this contradiction, studies to verify the distribution of Go alpha mRNA and protein in bovine and rat tissues were performed. Tissues were screened for the presence of Go alpha mRNA by use of a series of restriction fragments of a bovine retinal cDNA clone, lambda GO9, and oligonucleotide probes complementary to sequences specific among G alpha subunits for the 5' untranslated and coding regions of Go alpha. These probes hybridized predominantly with mRNA of 4.0 and 3.0 kb in bovine brain and retina. A 2.0-kb mRNA in retina also hybridized strongly with the cDNA but weakly with the oligonucleotide probes. In bovine lung, two mRNAs of 1.6 and 1.8 kb hybridized with the cDNA while only the 1.6-kb species hybridized with the coding-region oligonucleotide. In bovine heart, only a 4.0-kb mRNA was detected and in amounts much less than those in the other tissues. A similar distribution of Go alpha mRNAs was seen in rat tissues. In bovine tissues, Go alpha protein was identified with rabbit polyclonal antibodies directed against purified bovine brain Go alpha. An immunoreactive 39-kDa membrane protein was found principally in retina and brain, and in a lesser amount in heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of myosin light-chain kinase from bovine adrenal medulla.

Partially purified bovine adrenal medullary myosin light-chain kinase (MLCK) possesses a Stoke's radius of 79 A and a sedimentation coefficient of 3.95 +/- 0.45 S, yielding a native molecular weight of 150,000 +/- 17,000 g/mol and a frictional ratio of 2.24. It catalyzes the phosphorylation of the isolated light chain of skeletal muscle myosin and the light chain of intact adrenal medullary myosin, but not phosphorylase b or histone. The activation of MLCK by calmodulin is specific and dose dependent, yielding a K0.5 value of 9.0 nM; the dose response curve with respect to free Ca2+ is biphasic, exhibiting a stimulatory phase at low free Ca2+ concentrations (K0.5 = 0.17 microM) and an inhibitory phase at higher free Ca2+ concentrations (400-3000 microM). Michaelis-Menten kinetics are observed for ATP, yielding a Km for ATP of 25 microM and a Vmax of 23.2 nmol/min/mg. However, positive cooperative kinetics are observed for the skeletal muscle myosin light chain, yielding a Hill coefficient of 3.57, a K0.5 for light chain of 27 microM and a Vmax of 16.6 nmol/min/mg. A stoichiometry of phosphorylation of approximately 1 mol of phosphate/mol of skeletal muscle myosin light chain was observed. Therefore, adrenal medullary MLCK is similar in most, but not all, of its physical and kinetics properties to MLCKs isolated from other sources and may serve to regulate actin-myosin contractile activity in the adrenal medulla.

Factors affecting the use of influenza vaccine in the institutionalized elderly.

Annual influenza vaccination has been recommended for the past four years for all the patients residing on the extended-care facility of this large county hospital. During the fall of 1983, baseline data was collected regarding compliance with these recommendations. It was found that only 33% of the high-risk individuals had been vaccinated. A study was planned to investigate the factors for poor compliance rate and explore alternate methods of delivery of the vaccine. Despite the physician's strong belief in the vaccine, all methods of reminders to the physicians failed to have significant impact on increased use of the influenza vaccine. Finally an institutional policy pertaining to a standing order for the influenza vaccine was approved by the Infection Control Committee and the hospital Medical Staff Committee and 95% of the patients on the extended-care facility of this hospital were immunized.

Nosocomial infections among patients in a long-term care facility: spectrum, prevalence, and risk factors.

During a 6-month period data were collected on 460 patients residing on the long-term care division of this 1200-bed county hospital. The purpose was to determine prevalence and spectrum and to identify risk factors for skin infections, urinary tract infections, respiratory infections, and sepsis. Overall, the prevalence of nosocomial infections among 460 patients was 12%. Patients with infections had an average of 2.8 diagnoses of their underlying disease compared to patients without infections, who only had 1.8 diagnoses. Specific risk factors were identified. Skin infections were more common in patients who were nonambulatory, diabetic, malnourished, and incontinent of urine and feces, whereas respiratory infections were more common in patients who were smokers or had chronic obstructive lung disease and had not received pneumococcal vaccine. Thirteen percent of patients with an indwelling urinary catheter had symptomatic urinary tract infections, whereas 100% had asymptomatic bacteriuria.

Bacteremia in a long-term care facility. Spectrum and mortality.

One hundred episodes of bacteremia were studied in a primarily geriatric population. Gram-positive bacteremia accounted for 24% of all bacteremia (50% mortality rate), while gram-negative bacteremia accounted for 67% of bacteremia (25% mortality); 9% of all bacteremias were polymicrobial in nature (67% mortality). Overall mortality was 35%. The urinary tract was the most frequently identified tissue source (56%) followed by skin and subcutaneous tissue (14%) and respiratory tract (10%). Escherichia coli, Proteus species, and Klebsiella enterobacter group were the most common gram-negative organisms, Staphylococcus aureus was the most common gram-positive organism and together they accounted for approximately 75% of all bacteremia. Fifty percent of deaths occurred within 24 hours of diagnosis of bacteremia, despite appropriate antibiotic therapy. This study may help to identify risk factors for bacteremia in elderly patients.

Structure of polyadenylic acid in the ribonucleic acid of Saccharomyces cerevisiae.

Investigations of the structure of polyadenylic acid [poly(A)] in yeast have shown that there are two classes of poly(A) distinguished by size and kinetics of synthesis. Each class is found directly on the 3' end of messenger RNA. One class contains poly(A) molecules ranging from 60 to less than 20 nucleotides long. The longest molecules in this poly(A) class are the first to become labeled when cells are exposed to [3H]adenine. Label then appears in progressively smaller molecules. The second class of poly(A) is about 20 nucleotides long. The length homogeneity of this class and the presence in nuclear DNA of many copies of a polythymidylate sequence which is the same length suggests that this poly(A) is synthesized by transcription from DNA.