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Although these are simple principles purchase terazosin 1 mg free shipping heart attack vegas, they should not be forgotten in the modern profusion of technology-based monitoring methods purchase terazosin pulse pressure how to calculate. Chest radiograph findings include cardiomegaly order 1mg terazosin fast delivery heart attack heart attack, pulmonary edema, or alternately, a paucity of pulmonary vascular markings associated with inadequate pulmonary blood flow. Laboratory data include an increasing calculated base deficit or serum lactate or persistent lactate above 2 mmol/L (29,30). With the caveat that troponin is elevated after any intracardiac surgery, troponin levels that do not decrease after surgery have been associated with higher risk of poor outcomes. These include residual anatomic defects such as outflow tract obstruction, presence and degree of valvar regurgitation or stenosis, pericardial fluid or thrombus leading to a diagnosis of tamponade, assessment of right ventricular function, tricuspid regurgitation jet and position of the interventricular septum to assess pulmonary hypertension, and many others (33). If standard transthoracic views are not adequate for any reason, transesophageal echocardiography can be performed in the intubated, sedated patient (34). The growing availability of portable, lightweight bedside ultrasound systems that have good image quality for cardiac structure and function has made this tool indispensible in modern critical care. The immediate availability of echocardiography, inclusion of bedside ultrasound in residency and fellowship curricula, and the ability to save images and echo clips have all contributed to the rapid increase in the use of this modality (35). Obviously this technique is used during cardiac catheterization for calculation of cardiac output using the Fick method. A measured, not calculated, oxygen saturation, or ScvO , can be an2 important trend monitor to assess cardiac output and oxygen delivery. This measurement can be made intermittently, or during changes in patient status, or in response to interventions. Continuous monitoring of intravascular oxyhemoglobin saturation using reflectance catheters has been used in the umbilical artery, pulmonary artery, and adult-sized central venous catheters for a number of years, but only recently have standard pediatric-sized catheters become available for routine use to measure central venous oxygen saturation (ScvO ) in pediatric patients. In several small series of pediatric patients undergoing cardiac2 2 surgery, good correlation between ScvO as measured with the catheter versus blood co-oximetry (2 r = 0. Although assessment of each patient must be individualized, ScvO below2 50% in patients with univentricular hearts is often associated with low systemic oxygen delivery. Goal-directed therapy targeting ScvO >50% is associated with high postoperative survival and low2 complication rate in this population (40). The level of evidence was deemed B, limited populations evaluated and data derived from nonrandomized studies (41). Most devices utilize 2 to 4 wavelengths of infrared light at 700 to 1,000 nm, where oxygenated and deoxygenated hemoglobin have distinct absorption spectra. Since its now classic description in 1977 by Jobsis (42), this technology has been the subject of over 1,000 publications, and because of its noninvasive compact, portable nature and potential to measure tissue oxygenation in the brain and other organ systems during surgery and critical illness, it is gaining more widespread clinical use. The strong physiologic rationale together with existing case series and cohort studies have provided sufficient evidence for many practitioners to adopt this monitoring as standard practice in these institutions. Cardiac index may be measured by standard thermodilution methods, with care taken to input the correct calculation constant into the monitor software according to the catheter size and length, and volume and temperature of injectate. The average of three consecutive injections made in rapid succession at the same point in the respiratory cycle, that is, expiration, will optimize conditions to achieve an accurate measurement during steady state conditions. There are data from adult and pediatric critical care literature suggesting that the ability to increase and maximize both oxygen delivery and consumption may improve outcome, and is a predictor of survival from critical illness, including postoperative cardiac surgery (57,58,59,60). The level of evidence, however, is C, limited populations studied consisting of case series, and consensus of experts (61). A dilute solution of lithium chloride is injected into the vein, and arterial blood is withdrawn into the lithium electrode. The cardiac index is related to the area under the curve of the change of lithium concentration. This method has been demonstrated to have reasonable correlation with thermodilution cardiac output in children after congenital heart surgery. Cold saline is injected into a central venous catheter, and via a thermistor placed in a femoral artery, a time temperature curve is derived, which correlates reasonably well with standard thermodilution cardiac output as measured by a standard pulmonary artery catheter (63). This continuous method is periodically calibrated using the transpulmonary thermodilution cardiac output as described above (again making the method invalid with intracardiac shunting), and demonstrates a good correlation with transpulmonary 2 thermodilution in a recent study of 24 pediatric patients after cardiac surgery (r = 0. A recent survey of 162 pediatric cardiologists, intensivists, and surgeons found that serial lactate monitoring was the monitoring strategy most used by respondents (94%) (69). The oxygen carrying capacity of blood is improved by increasing the hemoglobin concentration. Each of these factors should be adjusted for the specific congenital cardiac lesion and the cardiovascular physiology that is associated P. Pharmacologic Therapy for Congenital Heart Disease The goal of drug therapy in an acute setting should be to optimize cardiac output and oxygen delivery; improve perfusion pressure to vital organs such as brain, heart, and kidneys and maintain an optimal balance between systemic and pulmonary blood flows with an appropriate level of oxygenation. Therefore, it is important to understand the actions of pharmacologic agents and to use them judiciously at the lowest effective doses. Point A represents baseline end- diastolic volume on the pressure–volume loop on the left of each panel, and baseline stroke volume on the Starling curve on the left of each panel. With administration of intravascular volume, end-diastolic volume and stroke volume are augmented significantly from point A to point B. However, diastolic compliance is nonlinear, and increases in stroke volume are progressively less with more intravascular volume administration from points B to C, and C to D. At high end-diastolic volumes and pressures, pulmonary capillary leak ensues resulting in pulmonary edema and pleural effusions. Enhanced ventricular relaxation and compliance result in an increased end-diastolic volume at the same pressure, point A versus B. Slowing heart rate, or instituting an agent such as milrinone can improve lusitropy. Increases in the slopes of the pressure–volume loops from points A, to B, to C, represent progressively increased inotropic states. Increased contractility results in a higher stroke volume ejected at the same left atrial pressures. At normal inotropic state, represented by the changes from point A to point B, lowering afterload allows the heart to eject to a lower systolic pressure, resulting in a higher stroke volume. With a decreased baseline inotropic state, represented by the changes from point C to point D, the increase in stroke volume with afterload reduction is much greater for a comparable change in afterload. Heart failure patients, and neonates are particularly sensitive to changes in afterload. Inodilators (milrinone, levosimendan) The cardiac intensivist must keep in mind that the indications for and doses of these drugs in an individual patient are highly variable. Affects such as age, disease state, and adrenergic receptor up or downregulation necessitate frequent titration of drugs to effect. Inotropes Epinephrine Epinephrine is an endogenous catecholamine that is secreted by the adrenal glands and has strong alpha- and beta-adrenergic receptor activation. This action on both types of adrenergic receptors leads to the complexity of response in different organs and tissue beds. The response of exogenously administered epinephrine depends on the ratio of alpha- to beta-receptors in the individual tissue beds as well as to the dose of epinephrine given. Activation of the β2-receptors in the vasculature of the skeletal muscles usually leads to a decrease in the systemic vascular resistance and the diastolic pressure. As the dose is progressively increased, more prominent peripheral vasoconstriction is seen due to the activation of the α-receptors in other vascular beds (74).
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In the transethmoidal route the already identifed superior Two further indentations are made above this one thereby turbinate is used as the critical landmark for sphenoidot- measuring 12 mm from the bony rim of the posterior choana omy terazosin 1 mg cheap hypertension kidney disease. Once the turbinate is removed fush with the face of the sphenoid directly above the third indentation generic 5mg terazosin with visa 5 htp arrhythmia. The anterior face of the sphenoid buy terazosin 5 mg cheap blood pressure pulse 90, the microdebrider is used to tip of the instrument is pushed through the bone and once palpate the face for the natural ostium of the sphenoid. It it has penetrated the sphenoid cavity it is twisted to enlarge is usually found at the junction of the lower one-third and the opening. A microdebrider or Kerrison punch is then used upper two-thirds but may be as high as the halfway point of to further open the sinus. The natural ostium is opened with the superior turbinate and is usually medial to the turbinate the punch or microdebrider initially inferiorly toward the on the anterior face of the sphenoid6 (Fig. However, There should be at least 8 to 10 mm of anterior face of the in patients with a sphenoethmoidal (Onodi) cell, the natu- sphenoid still above this lateral opening and this newly cre- ral ostium may be lower and more medial as the sphenoid is ated opening should therefore be well below the optic nerve. If the tip of the microdebrider does not fall into the natural ostium, medially and inferiorly as well (Fig. The noid is present then the following measurement technique anterior face of the sphenoid is removed up to the skull base should be followed to open the sphenoid. The bony rim of and laterally to the lamina papyracea and then fnally lowered Fig. The lower one-third of the superior tur- the posterior ethmoids have not as yet been opened. Note rior ethmoids have been sequentially opened the superior turbinate is how the anterior face of the sphenoid sinus is contributed by the sphe- divided into the thirds. The postnasal artery or vertical If the superior turbinate has been completely or partially branch of the postnasal artery is often cut during widening removed by previous surgery, the measurement technique of the sphenoid and requires bipolar cautery (Fig. In described previously is used to locate the position of the patients with signifcant new bone formation, an inferiorly natural ostium of the sphenoid sinus. Resection of part of the based mucosal fap can be elevated and the underlying bone superior turbinate may possibly remove some olfactory neu- drilled away before the fap is replaced. Studies done on olfaction after this technique have not been shown to adversely afect the sense of smell of the patient after surgery. Complications of Sphenoidotomy Epistaxis Bleeding is very common from the posterior nasal artery as it traverses the anterior face of the sphenoid on its way to the posterior septum. The posterior nasal artery is a branch of the sphenopalatine artery and travels horizontally just above the posterior choana (Fig. If this vertical branch is transected (and it often is as the sphenoid ostium is enlarged) a minor bleeder will be seen on the lower opening of the sphenoid. If the sphenoid os- tium is further enlarged inferiorly toward the foor of the sphenoid sinus, the main trunk of the posterior nasal ar- tery can be transected and result in a signifcant bleeder which usually spurts in a horizontal and medial direction. Attempted entry into the sphenoid in the superior lateral region will take the surgeon onto the orbital apex and optic nerve and even into the anterior skull base. In general this situation begins when entry into the posterior ethmoids through the vertical portion of the ground lamella is made too high. The whole direction of surgery is then toward the skull base and if the surgeon is inexperienced and does not recognize this, he or she may assume that the structure in front of them is a sloping anterior wall of the sphenoid sinus rather than the sloping wall of the skull base. If the skull base is thinner than usual then entry into the anterior cra- nial fossa and/or damage to the optic nerve can occur. The key to preventing this devastating complication is to iden- tify the superior meatus as the frst step in the dissection of the posterior ethmoids immediately after the ground la- mella is penetrated. This branch runs along the anterior face of the sphenoid, and can be inad- is clearly identifed. If this dissection is continued and the vertently transected during sphenoidotomy. The other area that the optic nerve can be damaged is in the superolateral wall of the sphenoid sinus. The optic nerve is dehiscent in ,12%5 of patients and inadvertent injury during surgery within the sphenoid sinus may lead to visual loss or blindness. It is tempting to use the microdebrider to remove polyps in the sphenoid but great care needs to be taken using the microdebrider in this sinus. In general the microdebrider should only be used along the foor and medial wall of the sphenoid and not in the superior or lateral region. Surgeons are also concerned when the sphenoidotomy is widened in a lateral direction. There are a few rules to follow during sphe- noidotomy that can minimize the risk to the optic nerve. The natural ostium of the sphenoid is generally around the lower one-third of the superior turbinates’ insertion into the ante- rior face of the sphenoid. The forward-biting Hajek Kofer punch is used to enlarge this natural ostium frst inferiorly Fig. As the arrow) inserting into the anterior face of the left carotid artery (solid arrow). Rotation of this septum during removal may result in a tear of orbit is approached the instrument should be well below the the wall of the carotid artery. The second rule is that if the distal jaw of the biting mecha- nism of the Hajek Kofer punch can be placed behind a bony septation it is generally safe to remove. Even a septation arising from the optic nerve can be safely removed onto the nerve by This again emphasizes the risk of using a microdebrider allowing the punch to rest on the nerve while the septation is with the cutting gate toward the lateral wall of the sphe- engaged between the jaws of the punch and cleanly removed noid. It only takes milliseconds for a dehiscent carotid ar- from the surface of the nerve. However, the superolateral tery wall to be damaged by the very efective cutting mech- region of the anterior face of the sphenoid is very carefully re- anisms in microdebriders with potentially catastrophic moved to lessen any possible risk to the optic nerve (Fig. In some patients the natural ostium of the sphenoid sinus is very small and the surgeon is unable Damage to the Carotid Artery to identify it and thus allow a safe entry into the sphenoid sinus. If a small or narrow instrument is pushed hard onto The other major structure at risk during sphenoidotomy is the the face of the sphenoid in an attempt to penetrate into the carotid artery. The bony wall covering the carotid is in most sphenoid, the bone may suddenly give way with the instru- patients thin and dehiscent in 5 to 8%5,11 of patients (Fig. This instrument can then inadvertently penetrate the thin or dehiscent bone or dehiscent carotid with subsequent massive hemorrhage. In the operative technique previously described, a blunt Freer elevator is used to push through the anterior face of the sphenoid sinus. The entry into the sphenoid can be well controlled with this instrument and the surgeon can easily feel as the bone is penetrated with- out risk of the instrument slipping inadvertently into the sinus. Also the instrument has a large blunt surface and the penetration point is medial and inferior and is thus less likely to injure the carotid. The second situation where a carotid injury may occur is if the intersinus septum of the sphenoid is attached to the anterior face of the carotid artery (Fig.
However buy discount terazosin line hypertension vs hypotension, patients of adrenal incidentaloma with subclinical Cushing’s syndrome should be considered for surgery if age is <40 years with recent onset/worsening of diabe- tes terazosin 5mg generic blood pressure medication iso, hypertension discount terazosin online hypertension uncontrolled icd 9, or osteoporosis. If aldosterone excess is established and age is >40 years, bilateral adrenal venous sampling is indicated. This is because of the fact that the incidence of adrenal incidentaloma increases with advancing age, and apparently normal looking contralateral adrenal gland may be functionally abnormal. Unilateral nonfunctioning adrenal incidentaloma with size >4 cm or imaging phenotype suggestive of adrenocortical carcinoma irrespective of size should be 4 Cushing’s Syndrome: Clinical Perspectives 93 operated. Those with unilateral nonfunctioning adrenal incidentaloma <3 cm should undergo biochemical and radiological imaging annually, and a growth in size >1 cm/year or development of hyperfunction should undergo operative removal. Bilateral nonfunctioning adrenal incidentaloma after careful exclusion of pheochromocytoma should undergo fine-needle aspiration cytology to estab- lish the diagnosis and be treated accordingly. The causes of bilateral adrenomegaly with adrenal insufficiency include infec- tions (e. The figures given below illustrate a patient who presented with adrenal insufficiency and bilateral adrenal masses and was found to have adre- nal histoplasmosis on laparoscopic adrenal biopsy. Cortisol secretion peaks at 0400–0800h and troughs at 2300–2400h, and this diurnal rhythm is established by 2–3 years of age. Diurnal variation of cortisol secretion prevents sustained hypercortisolemia, which may be detrimental to neuronal function and sleep. Loss of diurnal rhythm of cortisol secretion is defined as a 1600h serum cortisol level more than 50% of 0800h serum cortisol or 2300h serum cortisol >207 nmol/L. This is the earliest abnormality of the hypothalamo–pituitary–adrenal axis in patients with Cushing’s syndrome. Other causes of altered diurnal rhythm include pseudo-Cushing’s syndrome, seizure disorder, depression, use of anticonvulsants, and shift workers. Pregnancy and glucocorticoid resistance syndrome are associated with pre- served diurnal rhythm of cortisol secretion despite high serum cortisol. Liddle’s protocol was first described in 1960 and is also called as “adrenal sup- pression tests. Dexamethasone is the most potent glucocorticoid in suppressing hypothalamo– pituitary–adrenal axis (17 times more potent than hydrocortisone). In addition, it has no cross-reactivity to hydrocortisone and other metabolites; therefore, it does not interfere with cortisol assay. Hence, dexa- methasone is the preferred glucocorticoid for cortisol dynamic tests. The morning cortisol between 0800 and 0900h is helpful in differentiating exogenous Cushing’s from endogenous Cushing’s syndrome, as it is sup- pressed in patients with exogenous Cushing’s syndrome. This is important because exogenous glucocorticoid administration is the most common cause of Cushing’s syndrome. The other utility of morning cortisol is to establish the loss of circadian rhythm as compared to evening (1600h) cor- tisol. Exogenous Cushing’s syndrome is usually associated with 0800h cortisol <100 nmol/L. A detailed history and examination revealed that the patient did not have any discriminatory feature of Cushing’s syndrome, and hence a routine screening was unwarranted. Out of these three, any one test can be performed initially and if positive needs subsequent confirmation with one of the remaining two tests. Midnight serum cortisol estimation can be used as an alternative screening test for the diagnosis of Cushing’s syndrome in certain situations. An approach to a patient with suspected Cushing’s syndrome is depicted in the figure given below. What is the importance of midnight serum cortisol estimation in the diag- nosis of Cushing’s syndrome? Loss of circadian rhythm is the earliest biochemical abnormality in the evolu- tion of Cushing’s syndrome; thus, estimation of midnight serum cortisol may be used as an alternative screening test for the diagnosis of Cushing’s syndrome in certain situations. Late-night salivary cortisol could have been an alternative in place of midnight serum cortisol in the above mentioned situations, but the data is scanty. What is the sensitivity and specificity of various screening tests for the diagnosis of Cushing’s syndrome? The sensitivity and specificity of different screening tests for the diagnosis of Cushing’s syndrome are summarized in the table given below. What are the minimum tests required to establish the diagnosis of Cushing’s syndrome in a resource constraint setting? A 0800h cortisol does not have a discriminatory value, but it is important to rule out exogenous Cushing’s syndrome. The differentiation of pseudo-Cushing’s from Cushing’s syndrome is a clinical challenge as both share many clinical as well as biochemical similarities. Ideally, a patient of pseudo-Cushing’s syndrome with known inciting cause like alcohol, or depression should be reevaluated, after resolution of primary disorder. However, in clinical scenario it is often impractical; hence, we need definite diagnostic tests to differentiate pseudo-Cushing’s syndrome from Cushing’s syndrome. However, none of these tests have 100% specificity; hence, prospective follow-up is the best tool to discriminate between the two disorders. The tests which help in the differentiation of pseudo-Cushing’s syndrome from Cushing’s syndrome are summarized in the table given below. What are the tests available for establishing the etiological diagnosis of Cushing ’ s syndrome? The measurement of 0800h cortisol helps in excluding exogenous Cushing’s syndrome. The approach to a patient with Cushing’s syndrome is summarized in the figure given below. Approximately 90% of circulating cortisol is in bound form (cortisol-binding globulin and albumin), while the rest is in free form, and this fraction is freely filtered across glomerulus. Ninety percent of filtered cortisol is reabsorbed in renal tubules, and the rest is excreted in urine. In hypercortisolic states, the frac- tion of serum free cortisol increases, with consequent increase in urinary corti- sol excretion. Patients with cyclical Cushing’s syndrome exhibit periodic hormonogenesis, and “cycle” may vary from days to months; therefore, repeated measurements over a long duration are required for detection of hypercortisolemia. A 35-year-old woman with suspicion of Cushing’s syndrome has urinary free cortisol of 150 μg/day. A 40-year-old man with suspicion of Cushing’s syndrome has late-night salivary cortisol of 8 nmol/L. Poor oral hygiene, smoking and tobacco chewing may falsely elevate the late- night salivary cortisol; hence prior to estimation of late-night salivary cortisol estimation, it must be ensured that the patient has good oral hygiene and does not chew tobacco or smokes. Liquid chromatography – mass spectrophotom- etry is the best available assay; however, enzyme-linked immunosorbent assay is more commonly available and is equally sensitive. A detailed history revealed that the index patient was a betel nut chewer, there- fore, other screening tests should be advised for evaluation of Cushing’s syndrome. A 35-year-old female, working in a call center on night shifts presented with specific features of Cushing’s syndrome. But for practical purposes, they can be evaluated after stabilization of work schedule, with sleeping time considered as night and awake time considered as day.
Under these conditions buy terazosin 5 mg overnight delivery arrhythmia 101, other contaminants will not bind to the matrix and can be washed away discount 1mg terazosin overnight delivery arrhythmia during exercise. Escherichia coli order cheap terazosin blood pressure chart symptoms, named for the German physician Theodor Escherich (1857–1911), is a gram-negative, rod shaped bacterium propelled by long, rapidly rotating flagella (Figure 3. It is part of the normal flora of the human mouth and gut, helping to protect the intestinal tract from bacterial infection, aiding digestion and producing small amounts of vitamins B12 and K. The bacterium, which is also found in soil and water, is widely used in laboratory research and is probably the most thoroughly studied life form. A cut-away model of the bacterium showing some of the cellular layers and components, and E. As far back as the mid-1940s it was known that bacterial cells were able to exchange genetic material with each other in a semi- sexual manner. The experiments of Lederberg and Tatum clearly demonstrated the transfer of genetic information through bacterial conjugation (Lederberg and Tatum, 1946). The ability to perform this transfer is conferred by a set of genes called F (for fertility). When conjugation occurs, the F genes start travelling across the pilus, dragging the rest of the genome behind them. The bacterial genome can be measured, in minutes, from the origin of transfer with the amount of time it takes for a particular gene to be transferred from one bacterium to another indicating how far it is from the origin of replication. However, the size of the F episome precludes easy analysis and manipulation and its gene transfer properties can make it unstable. Most commonly used vectors are based either upon plasmids or bacteriophage lambda (λ). In general, vectors can be thought of as a series of discrete modules that provide requirements essential for efficient molecular cloning. A huge array of different types of vector is available today, with many being highly specialized and designed to perform a specific function. In this chapter, I will discuss some of the general points of vector design, but will concentrate on vectors that are commonly used in cloning experiments (Table 3. Plasmids are widely distributed throughout prokaryotes and range in size from approximately 1500 bp to over 300 kbp. The replication of the plasmid is often coupled to that of the host cell in which it is maintained, with plasmid replication occurring at the same time as the host genome is replicated. Plasmids are often described as being either relaxed or stringent on the basis of the number of copies of the plasmid that are maintained within the cell. At least part of the basis of this difference is the different mechanisms employed by plasmids in order to replicate themselves. In general, relaxed plasmids replicate using host derived proteins, while stringent plasmids encode protein factors that are necessary for their own replication. Other gene names reflect biological function, for example, Hbb for the haemoglobin β-chain, and Adh for alcohol dehydrogenase enzymatic activity. Drosophila geneticists have brought the most whimsical approach gene naming – with names like fushi tarazu (ftz, from the Japanese words meaning ‘segment deficient’), spatzle¨ (spz, a type of German noodle), dunce (dnc), forkhead (fkh), hedgehog (hh)andether-a-go-go (eag). The assignment of chromosomal locations for genes of unknown function developed soon after the establishment of successful metaphase spreads, chromosome banding methodologies, somatic cell hybrids, isozyme separation and the ability to associate genes and phenotypes with a particular site on the chromosome. It has also become common to use the same name for the gene as for the enzyme or other protein that it encodes. Often, the gene name is italicized whereas the gene product is not to distinguish between the two. This can, however, be a source of confusion, since there is not necessarily a one-to-one relationship between the two entities. Additionally, nomenclature used for one organism or species may be different in another. Traditionally, recombinant plasmids tend to bear the initials of their creator(s) followed by a number that may indicate the numerical order in which the plasmids were produced, or perhaps has some deeper meaning. Other plasmids are named for specific, but still comparatively obscure, reasons, e. Although many attempts have been made to standardize nomenclature (White, Mattais and Nebert, 1998), historical names tend to be maintained. Most plasmids in common use today are based upon the replication origin of the naturally occurring E. Colicin E1 is a transmembrane protein that causes lethal membrane depolarization in bacteria (Konisky and Tokuda, 1979). The drug resistance gene codes for a protein that interferes with the action of colicin by inhibiting its ability to form a channel through the bacterial membrane (Zhang and Cramer, 1993). Bacteria harbouring the ColE1 plasmid can be distinguished from their counterparts that do not possess the plasmid by their ability to grow on plates containing colicin E1. Unlike the bacterial origin of replication (OriC), however, the replication of ColE1 proceeds in one direction only. It codes for, amongst other things, the bacteriocin colicin E1 (the product of the cea gene) and an immunity protein (the product of the imm gene) that prevents the toxic effects of the bacteriocin in cells harbouring the plasmid. The relative positions of these replication control elements to the origin (ori)are shown. The arrangement of regulators results in plasmid incom- patibility – that is, two plasmids with the same origin of replication cannot coexist in the same cell. This is impor- tant for cloning experiments when a mixed population of plasmids, which is often the result of a ligation reaction, is transformed into bacteria. Individual transformants produced in this way will contain a single plasmid and not a mixed population. What was required was a plasmid that could be replicated in the same way as ColE1, but in which recombinants could be easily recognized. This plasmid contains the ColE1 origin of replication (ori and rop), together with two antibiotic resistance genes. Transformed cells are first grown on bacterial plates containing ampicillin to kill all the cells that do not contain a plasmid. Those cells that grow on ampicillin are then replica plated onto medium containing both ampicillin and tetracycline. Embedded within the coding sequence of lacZ are the recognition sites for a number of restriction enzymes. Certain mutations in the 5 region of lacZ prevent subunit association of the resul- tant protein (ω-peptide) and the monomers lack enzyme activity (Ullmann, 1992). In some such mutants, subunit assembly (and enzyme activity) can be restored by the presence of a small (50- or so amino-acid) amino-terminal fragment of the lacZ product (the α-polypeptide) (Juers et al. The product of the lacZ M15 allele lacks amino acids 11–41 of wild-type β-galactosidase and is subject to α-complementation. The indoxyl spontaneously dimerizes and oxidizes to form an insoluble blue dye (5,5 -dibromo-4,4 -dichloro-indigo; not shown). The β- galactosidase enzyme is the product of the lacZgeneandtheactiveformoftheenzymeis a tetramer of identical polypeptides. Certain mutants of lacZ(suchaslacZ M15) produce versions of the protein that do not include the extreme amino-terminal end of the 1173 amino acid polypeptide.
