By A. Mirzo. South Dakota State University.

Medially purchase antabuse online, the ligaments form a continuous Connection longitudinal attachment to the pia mater purchase cheapest antabuse. Laterally discount antabuse 500 mg with amex, they form triangular buy genuine antabuse on line, toothlike processes that attach The internal vertebral venous to the dura. Because of their pial attachments mid- plexus forms a valveless com- way between the posterior and anterior surfaces of munication between the cranial dural sinuses, the spinal cord, the denticulate ligaments can be which collect blood from the veins of the brain, used as landmarks for surgical procedures. The spi- and the veins of the thoracic, abdominal, and nal cord is also anchored by the roots of the spinal pelvic cavities. It, therefore, provides a direct nerves, which are ensheathed by a cuff of dura where path for the spread of infections, emboli, or they perforate it near the intervertebral foramina. Dura Mater Inferior or caudal to the spinal cord, the dura The spinal dura mater loosely surrounds the spinal mater forms thedural sac(Fig. The area between the spinal dura and the inferiorly to the middle third of the second sacral periosteum lining the vertebral canal is the epidural vertebra. Chapter 2 Spinal Cord: Topography and Functional Levels 21 flum terminale, the threadlike extension of the descending course within the subarachnoid space pia mater, and descends to the back of the coccyx (Fig. Therefore, the dural sac nerves, which then exit from the intervertebral contains (1) the flum terminale; (2) the cauda foramina and immediately begin to branch. A hypodermic needle may be introduced nent of these is the anterior median fssure, occu- into the subarachnoid space (Fig. On the opposite side the spinal cord, thereby causing irreparable dam- is a far less conspicuous groove, the posterior age, because regeneration or repair to neurons and median sulcus. The anterior and posterior root- axons in the spinal cord (or brain) does not occur. A large number of the fbers are puncture is contraindicated in patients with myelinated, thus accounting for the white color elevated intracranial pressure due to trauma, in the fresh or unstained state. White Matter Thus, each segment gives rise to four separate roots, one posterior and one anterior on each The white matter is divided into three areas, called side. According to their positions, these are to the spinal cord by a series of rootlets. The the posterior funiculus, the lateral funiculus, and posterior and anterior roots take a lateral and the anterior funiculus (Fig. Most of their neu- A well-defned separation between these two tracts rons play roles in voluntary movement, and many is not always evident. This is generally true of most of them give rise to axons that emerge in the of the tracts in the spinal cord; hence, the locations anterior roots. Hence, the anterior horns are pri- of the various tracts in the spinal white matter are marily the “motor” parts of the spinal gray matter. The intermedi- ate zones are composed mainly of association or The gray matter is divided into four main parts: interneurons for segmental and intersegmental 1. The anterior or ventral horns intermediate zones are the “association” parts of 3. The lateral horns ing from their neurons remain in the spinal cord; some, however, do project to the brain. For descriptive purposes, an imaginary hori- The lateral horn is a small triangular exten- zontal line passing from side to side through sion of the intermediate zone into the lateral the deepest part of each posterior funiculus and funiculus of the thoracic and the upper two lum- extending laterally through the gray matter bar segments. It contains cell bodies of pregangli- defnes the anterior boundary of the posterior onic neurons of the sympathetic nervous system. The posterior horns contain groups of neurons that are infuenced mainly Nuclei or Cell Columns by impulses entering the spinal cord via the posterior roots. Hence, the posterior horns are The neurons of the spinal gray matter are primarily the “sensory” parts of the spinal gray arranged in longitudinal groups of functionally matter, and many of their neurons give rise to similar cells referred to as columns or nuclei Chapter 2 Spinal Cord: Topography and Functional Levels 23 (Fig. For example, Myelin-stained transverse sections of the four the substantia gelatinosa and the proper sensory major regions of the spinal cord can be distin- nucleus, which are related to pain impulses from guished from each other most readily by the size all spinal nerves, extend throughout the length and shape of the respective gray matter (Figs. Because of the large size of the lower dorsal nucleus and the intermediolateral nucleus, limbs, the lumbar and sacral segments have mas- which are related to the cerebellar and auto- sive posterior and anterior horns. In lumbar seg- nomic systems, respectively, exist only in certain ments, the anterior horn has a distinct medial spinal cord segments. In addition, the Laminae rim of white matter surrounding the sacral gray The spinal gray matter can also be divided into matter is much thinner than that in the lumbar laminae or layers based on layerings of morpho- spinal cord. Laminae The posterior horn in both thoracic and cervi- provide a more precise identifcation of areas cal segments is narrow compared with lumbar and within the spinal gray matter and are very use- sacral segments. However, owing to the muscular ful in describing the locations of the origins or volume of the upper limbs, the cervical anterior terminations of the functional paths. Ten lami- horn is much larger than the thoracic, which nae make up the spinal gray matter, and, in mainly supplies the relatively small intercostal general, they are numbered from posterior to and subcostal muscles. Nevertheless, Lamina X is in the commissural area surrounding because the white matter contains axons trans- the central canal. Posterior median sulcus Posterior funiculus Posterior horn Substantia Intermediate gelatinosa zone Central canal Lateral horn Lateral funiculus Anterior horn Anterior funiculus Anterior median fissure Figure 2-7 Transverse section of thoracic spinal cord. Note the slim anterior and posterior horns and lateral horn indenting the lateral funiculus. Chapter 2 Spinal Cord: Topography and Functional Levels 25 Gracile tract Posterior median sulcus Posterior funiculus Cuneate tract Posterior intermediate sulcus Posterior horn Substantia gelatinosa Intermediate zone Lateral funiculus Anterior horn Anterior funiculus Anterior median fissure Figure 2-8 Transverse section of cervical enlargement. Note the slim posterior horn, the large anterior horn, and the division of the huge posterior funiculus. This is the anatomi- Injury to the spinal cord can be two fundamen- cal basis underlying sacral sparing (See Fig. Acute spi- nal cord injury can result from trauma or stroke, while chronic injury can result from infections, Chapter Review infammation, tumors, genetic disorders, and com- pression. Traumatic injury with momentary or Questions prolonged compression of the spinal cord has an immediate onset of clinical signs that vary depend- 2-1. What are the contents of the spinal ing upon the specifc tracts and neurons/nuclei epidural space? At what three intervertebral articulations are bination of trauma and vascular interruption comes dislocations most likely to occur and what with contusions to the spinal cord. What are the distinguishing characteristics white matter is more resistant to hypoxia than the of transverse spinal cord sections at sacral, gray matter. Each of the following concerning the cauda anatomical landmarks for: equina is true except one: a. The disparity between spinal cord levels spinal cord and vertebral levels in adults is due to b. Differential growth or elongation of the of the cord spinal cord compared to the vertebral c. The diminished size of the caudal spinal eral white matter may remain functional cord compared to rostral levels. It also contains the long tracts that transmit somatosensory impulses from all parts of the body to the forebrain, as well as motor impulses for vol- untary movements that originate in the forebrain. Damage to the brainstem is manifested by somatosensory or motor dysfunctions or both, accompanied by abnormalities in cranial nerve functions. The level of damage in a brainstem lesion can usually be determined by cranial nerve malfunction. Because of the vital nature of many functional centers located in the brainstem, especially at more caudal levels, brainstem lesions are frequently fatal. The brainstem is the stalk-like part of the brain The brainstem is covered posteriorly by the that is located in the posterior cranial fossa. It cerebellum to which it is connected by huge consists of the medulla oblongata, pons, and masses of nerve fbers that form the three pairs of midbrain (Fig. Its anterior surface is closely continuous with the spinal cord at the foramen related to the clivus, the downward sloping basal magnum, and the midbrain is continuous with surface of the posterior cranial fossa between the the forebrain at the tentorial notch, the opening dorsum sellae and foramen magnum (Fig. The posterior surface of its rostral stance, the brainstem is thrust downward as part is anatomically related to the cerebellum to the overlying mass of the cerebellum herni- which it is connected by the inferior cerebellar ates through the foramen magnum against the peduncles. Pressure on cardiovascular The caudal half of the medulla contains a and respiratory centers in the medulla quickly prolongation of the central canal of the spinal results in death. Corpus callosum Cerebral Fornix hemisphere Septum pellucidum Diencephalon Third ventricle Middle cranial Midbrain fossa Cerebral aqueduct Anterior cranial Cerebellum fossa Fourth ventricle Drosum sellae Pons Posterior cranial fossa Medulla Clivus Posterior margin of foramen magnum Spinal cord Figure 3-2 Magnetic resonance image of median view of right half of brain. Chapter 3 Brainstem: Topography and Functional Levels 29 of the medulla forms the caudal or medullary part are necessary to distinguish the subdivisions and of the foor of the fourth ventricle, the cerebrospi- their functional levels, these alone are described nal fuid–flled cavity between the cerebellum and here. Lateral to the rostral Pons part of each pyramid is a prominent elevation, The pons extends from the medulla to the mid- the olive. The sul- the cerebellum to which it is attached by the cus posterior to the olive is the postolivary sulcus, middle cerebellar peduncles or brachii pontis.

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It is also possible to pass a sucker through perature is less than 15°C buy generic antabuse pills, the ascending aorta is clamped the homograft valve to vent the left heart if there is evidence and cardioplegia solution is infused into the truncal root cheap antabuse online amex, of left heart distention proven antabuse 500mg. Distention of the homograft is a use- while the ductus is controlled with forceps buy antabuse 500 mg free shipping. Upon diffcult to distinguish ductal tissue, but if this is apparent it completion of the anastomosis, air is displaced by running should be excised. The remainder of the proce- the descending aorta to the site of excision of the pulmonary dure can be undertaken as for a standard repair. The arch anasto- is severely hypoplastic and will be inadequate to carry the mosis will be too proximal on the truncus and will interfere entire cardiac output. Instead, a longi- helpful to plasty the hypoplastic ascending aorta with a patch tudinal anastomosis should be made on the ascending aorta of autologous pericardium that extends from the proximal immediately proximal to the takeoff of the head vessels and perhaps extending a little on to the left common carotid artery truncal root to a point just beyond the anastomosis to the if this is a type B interruption (Fig. An alternative approach could aorta can be controlled with a C-clamp in order to reduce be to use a short segment of nonvalved femoral vein homo- tension on the anastomosis as this is fashioned. Tourniquets are no longer used on the pulmonary arteries to the proximal descending aorta. Because of the marked disparity between the proximal truncus and the distal ascending aorta, it is necessary to reduce the size of the proximal truncus by taking a tuck on the rightward and posterior aspect of the truncal root, thereby creating a dog ear. The ascending aorta is sutured to the left side of the truncal root which improves the lie of the homograft conduit. However, the patients who had more aggressive techniques were doing well at follow-up. The age who underwent primary repair of truncus arteriosus at median age at surgery was 2 weeks. Nine of the patients University of California, San Francisco between 1992 and had associated interrupted arch. A total of 23% of patients had moderate or severe trun- truncal valve regurgitation was diagnosed preoperatively, cal valve regurgitation and 12% had interrupted aortic arch. Five patients underwent months, there were two deaths resulting in a Kaplan–Meier truncal valve repair and one underwent homograft replace- estimate of survival at 1 year of 92%. The nifcantly associated with poorer survival over time were actuarial survival overall was 96% at 30 days, 1 and 3 years. The two deaths in the series occurred in patients replacement among early survivors was 57% at 3 years. None of the patients required with interrupted aortic arch and truncus at Royal Children’s reoperation because of truncal valve problems or aortic arch Hospital, Melbourne. Freedom from aortic reop- duit replacement was necessary in 17 patients after a mean eration was 76. Functional had an aortic homograft was 4 years and for those who had a status in all patients was good. Repeat surgical intervention truncal valvuloplasty methods has neutralized the traditional was rare and major complications related to root dilation did risk factor of truncal valve regurgitation. In this earlier timeframe, cal repair with a homograft conduit at Children’s Hospital truncal valve regurgitation, interrupted aortic arch, coronary Boston between 1990 and 1995. Although the early mortality artery anomalies, and age at repair greater than 100 days was satisfactory at 4. Pulmonary hyper- interrupted aortic arch or absent pulmonary valve syndrome, tensive episodes were fewer and duration of ventilator depen- the durability of homografts was disappointing. A total of dence, as well as absolute pulmonary artery pressure, were 47% of the patients underwent conduit replacement after a signifcantly less in patients undergoing correction before 30 mean follow-up of 34 months. Univariate analysis revealed that patients Other centers have confrmed the effectiveness of aggres- with a homograft with an internal diameter of 8 mm or less sive truncal valve repair, although initial truncal valve regur- were most likely to suffer early graft failure and reoperation. Simple cases had improved long- In 2001, Mavroudis and Backer26 described the results term survival relative to complex cases. Age, weight, pathol- of surgery for eight patients who underwent intervention for ogy, type of homograft (aortic versus pulmonary), length of severe truncal valve insuffciency between 1995 and 2000. Among the three report has suggested that failure of homografts in infants 582 Comprehensive Surgical Management of Congenital Heart Disease, Second Edition is not simply related to somatic outgrowth. The anatomy of common aor- reviewed imaging studies of 40 patients who had undergone ticopulmonary trunk (truncus arteriosus communis) and its embryonic implications. This is most commonly tion between Foxd3 and Pax3 in cardiac neural crest develop- observed at the annular area. Persistent truncus arteriosus: a clas- A number of authors have described the use of conduits sifcation according to anatomic types. Coronary arte- years in conduits larger than 16 mm at the time of implanta- rial and sinusal anatomy in hearts with a common arterial tion. A total of 23 of these patients had direct anastomosis Thorac Cardiovasc Surg 1992;104:1728–42. Coronary arteries in with a monocusp in 15 patients or a simple pericardial patch truncus arteriosus. Clinical angiographic and pathologic fndings conduit (28 patients) or a xenograft containing conduit (10 in 100 patients. Repair of truncus arteriosus and anastomosis had an 89 ± 10% freedom from reintervention interrupted aortic arch. They found that the cal valve and associated interrupted arch in neonates with trun- use of a xenograft conduit was an independent risk factor for cus arteriosus. For exam- 102 cases of complete repair of congenital heart defects in ple, Powell et al. Repair of trun- arial freedom from reoperation was 65% at 30 months after cus arteriosus in the neonate. True truncus arterio- resulted in signifcant immediate hemodynamic and angio- sus. Ann lation where conduit failure is predominantly secondary to Thorac Surg 1974;18:122–37. J Am cus arteriosus associated with interrupted aortic arch: long- Coll Cardiol 1997;30:1067–71. Truncal valve repair: initial fow tract reconstruction with bicuspid valved polytetrafuoro- experience with infants and children. Trends in with a bicuspid polytetrafuoroethylene pulmonary valve in the management of truncal valve insuffciency. Ann Thorac 41 children and adults: a new option for right ventricular out- Surg 1998;65:517–24. Eur J Cardiothorac Surg Improving early and intermediate results of truncus arteriosus 2001;20:95–103. Neonatal after neonatal or early infant repair of truncus arteriosus using repair of truncus arteriosus: continuing improvement in out- homograft conduits. In the past, this anomaly was there is fusion of the right and left sixth dorsal aortic arch thought of as being part of a spectrum with tetralogy with with the plexus of systemic arteries carried by the lung buds pulmonary stenosis and would have been described in the from the primitive foregut. Although there is no question that the two enti- process of fusion, pulmonary atresia results and there is a ties share a common embryological background and are part persistence of connections from the aorta. Even with optimal management, participate in septation of the common outfow tract into sep- these children require so many interventions that there is a arate aortic and pulmonary arteries. Indeed many would cells have been found to contribute to the adult semilunar consider that the quality of life for children at the severe end and atrioventricular valves, as well as part of the cardiac con- duction system. Certainly the total fnancial cost signifcantly higher in patients with pulmonary atresia with of treatment of these children is at the very far end of the multiple collaterals than those with simple tetralogy with entire spectrum of congenital heart disease. The lumen of the Duct dependent;Duct dependent; infundibulum may be very small and diffcult to locate. They are usually also in continuity with a vestigial main pulmonary artery that is also approximately 2 mm in diameter and usually extends to the atretic infundibulum (Fig. Although there is often excellent “arborization,” that is, distri- (c)(c) bution of the true pulmonary arteries to most of the broncho- pulmonary segments, there may on the other hand be limited distribution that will need to be defned. At the most mild end of the spectrum of tetralogy of Fallot with pulmonary atresia, the branch pulmonary arter- ies are normally developed and are supplied by a normally positioned ductus arteriosus. The main pulmonary artery may also be relatively well developed and extend to the atretic infundibulum. At the mildest end of the spectrum, there is even development of (d)(d) the pulmonary valve so that the patient may be considered to have “valvar” pulmonary atresia. Even fur- true pulmonary arteries and the presence of aortopulmonary col- ther across the spectrum, there is complete absence of devel- lateral vessels. In group 1, there is simple valvar or infundibular opment of the main pulmonary artery so that the right and atresia.

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The patent ductus venosus: an additional ultrasonic finding in portal hypertension purchase 250mg antabuse with visa. Persistent venous valves antabuse 250 mg line, maldevelopment of the right heart order cheap antabuse on line, and coronary artery-ventricular communications cheap antabuse 250 mg with amex. Cor triatriatum dexter: antemortem diagnosis in an adult by cross sectional echocardiography. Spinnaker formation of sinus venosus valve: case report of a fatal anomaly in a ten- year-old boy. Developmental aspects of the sinus valves and the sinus venosus septum of the right atrium in human embryos. Cor triatriatum dextran simulating right ventricular myxoma and pulmonary stenosis. Alomari Introduction Vascular anomalies are relatively common heterogenous disorders characterized by developmentally abnormal blood vessels including the venous, arterial, and lymphatic lineages. These anomalies may overlap clinically and radiologically creating considerable confusion in the clinical practice and published research. Nevertheless, the diagnosis and management of the vast majority of vascular anomalies fundamentally can be simplified if the proper nomenclature and classifications are applied. Commonly used inaccurate terms, such as lymphangioma, cystic hygroma, cavernous hemangioma, strawberry hemangioma, hemangiolymphangioma, and cavernoma should be abandoned for the more representative designation. The binary classification proposed by Mulliken and Glowacki in 1982 (1) divides vascular anomalies into two major categories: (1) vascular tumors and (2) vascular malformations. The correct diagnosis and appropriate therapy require proper awareness of the clinical and imaging features of different types of vascular anomalies. Improper terminology can lead to the wrong diagnosis, mistreatment, and misdirected research efforts (2). Unfortunately, terminology imprecision is still prevalent in the published literature. The authors also demonstrated that inaccurate designation of the vascular anomaly was associated with an increased risk of erroneous management. Embryology and Genetics The embryonic vascular network primarily is created via two major, distinct mechanisms: vasculogenesis and angiogenesis (4,5). The differentiation and growth of blood vessels from hemangioblasts of mesodermal origin to form the heart and the primitive vascular plexus is called “vasculogenesis,” while the subsequent process of remodeling and expanding this network is referred to as “angiogenesis” (6). The distinction between arteries and veins is an early developmental process (7) with molecular differences between arterial and venous channels prior to the establishment of the circulation (8,9). Due to the extensive, complex biologic pathways involved in the formation of normal blood vessels, developmental anomalies of the vasculature (both idiopathic and caused by specific defects) frequently are seen. Most vascular anomalies are sporadic with no identifiable familial or genetic predisposition. Nevertheless, specific mutations have been identified for many vascular anomalies and syndromes. Several sporadic, complex syndromes are known to be associated with fast-flow vascular anomalies. Classification The well-known classification of vascular anomalies, proposed by Mulliken and Glowacki (1), divided vascular anomalies into two distinct types: tumors and malformations. The differentiation is based on clinical, histopathologic and imaging differences (Table 37. The lesions are known to disproportionately affect Caucasians, females, and premature infants. Deep lesions affect the subcutis and adjacent anatomical spaces; typically sparing the skin and bone. Nevertheless, complications of hemangiomas, such as ulcers, bleeding, amblyopia (periorbital), airway obstruction (subglottic), and heart failure (liver) require prompt intervention (19,20). The raised, well-marginated reddish lesions have superficial (cutaneous) and deep portions. Congenital Hemangiomas Congenital hemangiomas are present and fully mature at birth. Liver hemangiomas represent a spectrum of lesions with variable clinical and imaging features. T2 and postcontrast T1 sequences demonstrate two focal, well-defined T2 hyperintense lesions that enhance following contrast administration. The tumor presents clinically with thickened, purple-ecchymotic skin discoloration (Fig. Imaging of Vascular Tumors Ultrasonography is the initial, simple and reliable imaging modality for many vascular tumors. Irregular patch of purple-ecchymotic skin thickening with overgrowth of the right thigh. A: Diffuse enlargement of the right parotid gland caused by an infantile hemangioma. Both modalities show well-defined, lobulated solid masses with homogeneous enhancement following contrast administration. Flow voids within or around the lesion are caused by the dilated feeding arteries and draining veins (Fig. Two well-defined, lobulated soft tissue masses in the left occipital and parapharyngeal spaces. Note the hyperintense signal and flow voids (enlarged vessels) on axial T2 sequence (A) and strong enhancement following contrast administration on axial T1 sequence (B). Large, solid hypervascular lesion of the right thigh with red-purple overlying skin. Congenital hemangioma usually displays a large, discoid cutaneous–subcutaneous well-defined mass with large feeding arteries and superficial vessels (Fig. Angiographic findings of congenital hemangioma include inhomogeneous parenchymal staining, large and irregular feeding arteries in disorganized patterns, arterial aneurysms, and direct arteriovenous shunts (29). Axial T2 sequence (A) shows skin thickening, reticular subcutaneous infiltrate and epifascial confluence of signal. If treatment is indicated, successful medical management typically is achieved using systemic corticosteroids or propranolol. Embolization of large arterial feeders and intratumoral shunts is reserved for hemangiomas causing high-flow heart failure. Pain can also be attributed to joint, muscle and tendon involvement and weakening. Recurrent hemarthrosis, particularly of the knee joint, is predisposed by synovial involvement and usually results in chronic pain and joint degenerative changes. This asymptomatic phenomenon is characterized by elevated d-dimer and hypofibrinogenemia with normal or slightly decreased platelet counts (32). Clots or phleboliths are typically incompressible, hyperechoic oval or round filling defects. Color and spectral Doppler interrogation typically shows no flow within the venous spaces and ultrasonography is invaluable to guide treatment. Intralesional or ascending venography is not typically needed as a diagnostic study. Venography shows amorphous, spongiform spaces with stagnant flow and variable types of draining veins (Fig. B: Partial, heterogenous enhancement on axial T1 sequence following contrast administration. The amorphous, spongiform communicating venous spaces are opacified with direct injection of contrast. Sclerotherapy is widely accepted as the initial treatment of choice at many specialized centers. Sclerotherapy, particularly for extensive lesions, is performed under general anesthesia. Intralesional placement is confirmed by free blood return and intralesional venography. It is crucial to implement radiation dose reduction techniques such as proper collimation, filtration, and last image hold. Contrast material should be diluted with saline without jeopardizing the imaging quality. After confirming the proper position of the needle, the lesion is injected with the sclerosant under fluoroscopic and/or sonographic guidance (Fig. Dual-needle technique is useful in focal lesions where two needles (or more) are simultaneously placed within the lesions. The sclerosant is injected through one needle displacing blood via the other (draining) needle.

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The column flow-through can be collected and will contain the purified target protein buy antabuse 250 mg overnight delivery. A variety of specific proteases have been used to cleave purification tags from target fusion proteins (Table 8 order 250 mg antabuse free shipping. For example cheap antabuse 500 mg amex, the protease Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile–Glu–Gly–Arg antabuse 250 mg low cost. However, it will sometimes cleave at other basic residues, depending on the conformation of the protein substrate, and a number of the secondary sites have been sequenced that show cleavage following Gly–Arg dipeptides (Quinlan, Moir and Stewart, 1989). Consequently, the protease may not only cleave the site between the tag and the target protein, but many also cleave the target protein itself. The recognition sequence of each protease is shown, together with the actual site of cleavage, depicted by the arrow Protease Recognition and Notes Reference cleavage site Factor Xa IleGluGlyArg↓ 42 kDa protein, composed (Nagai, Perutz and of two disulphide linked Poyart, 1985) chains, purified from bovine plasma Enterokinase AspAspAspAspLys↓ 26 kDa light chain of (LaVallie et al. The protein undergoes a large conformational change upon binding of maltose, and results in the formation of a stable complex (Figure 8. Bound proteins can be eluted from amylose by including maltose (10 mM) in the column buffer (Figure 8. The protein is depicted in a ribbon form with α-helices coloured in purple and β-sheets in blue. Inteins are a class of proteins, found in a wide variety of organisms, that excise themselves from a precursor protein and in the process ligate the flanking protein sequences (exteins) (Cooper and Stevens, 1995). The process of polypeptide cleavage and ligation is dependent on specific chemistry involving thiols and a conserved asparagine residue. Most inteins have a cysteine residue at their amino-terminal end and an asparagine at their carboxy-terminal end (Figure 8. All the information required for the splicing reaction is contained within the intein itself, and if these sequences are placed in the context of a target protein they still splice themselves out. The target gene is cloned into an expression vector such that a three-component fusion protein is produced, in which a target protein–intein–chitin binding domain fusion is produced. Chitin is a fibrous insoluble polysaccharide made of β-1,4- N-acetyl-D-glucosamine that is found in the cell walls of fungi and algae and in the exoskeletons of arthropods. This is a slow process and requires an overnight incubation to complete, which may prove problematical if the target protein is not stable under these conditions. The thioester is, however, unstable and will spontaneously hydrolyse to yield a native protein. Cysteine induced cleavage results in the insertion of a cysteine amino acid residue at the carboxy-terminal end of the cleaved polypeptide. Protein purification in these circumstances, if performed under suitably mild conditions, can lead to the isolation of naturally occurring protein complexes. They are associated, through non-covalent interactions, with a variety of other proteins that may be involved in the regulation of their function. The over-production of a single protein will not result in the over-production of other proteins in the complex. Therefore, to isolate complexes from cells, protein production should be as close to the natural state as possible. Cells containing the tagged protein are gently lysed and then applied to a column containing IgG, which binds with high affin- ity to Protein A. The two-step purification procedure is highly specific and can result in the isolation of contaminant-free protein complexes. The knowledge of the sequence of individual genes, and the entire genome, is vital if we are to understand not only how genes and proteins work but also how different gene products influence the activity of each other within the context of the whole organism. Here, we will dis- cuss a number of genetic and physical methods that have been used to map genomes. Our discussion will concentrate mainly on the mapping and sequenc- ing projects associated with the human genome, although readers should be aware that much of the groundwork for the elucidation of the human genome sequence has come from the analysis of other organisms – both prokaryotic and eukaryotic. The chromosome content of an organism (its karyotype) can be visualized using a microscope. By convention, the shorter arm of each chromosome is designated as p and the longer arm is designated as q. The different chromosomes of an organism are usually different sizes (ranging in the human from 279 × 106 bp for chro- 6 mosome 1 to 45 × 10 bp for chromosome 21), but most chromosomes are difficult to distinguish based on size alone by microscopy. Distinct chromo- some banding patterns can be obtained, however, when they are treated with certain dyes. Approximately 500 different bands can be obtained reproducibly after treating human chromosomes with the stain Giemsa (Figure 9. These banding patterns can be used to generate a cytological map of each chromo- some and provide a low-resolution mechanism to distinguish one portion of a chromosome from another. Some chromosome abnormalities that cause inher- ited genetic diseases can be observed by karyotype analysis – additional copies of chromosomes can be easily identified, e. For example, using some of the techniques described below, the gene mutated in sufferers of cystic fibrosis has been mapped to the long arm of chromosome 7 in banding region 31. The chromosomal location of the gene in the cytological map is therefore designated as 7q31. Metaphase chromosomes from a male were treated with the protease tryspin (to remove protein) and then stained with a mixture of dyes called Giemsa (named after Gustav Giemsa, who first used it) and viewed using a light microscope. Each pair of chromosomes has a similar length and banding pattern that allows them to be aligned. Chromosomes from a female would have two X chromosomes rather than the X and Y shown here 9. The first genetic map of a chromosome was constructed by Alfred Sturtevant usingdatafrom Drosophila mating crosses collected by Thomas Morgan (Morgan, 1910). Sturtevant used the frequency at which particular observable phenotypes were separated from other genes (through recombination events) during meiosis. The information gained from the experimental crosses could be used to plot out the location of genes – tightly linked genes are physically 9. Genetic map distances are based on crossover frequencies and are measured in centiMorgans (cM), while physical distances are measured in megabase pairs (Mbp) or kilobase pairs (kbp) located close to each other, while those that were only weakly linked are physically further apart. Sturtevant constructed a genetic map of the locations of six genes on the X chromosome of Drosophila melanogaster (Sturtevant, 1913). Many other gene traits in a variety of different organisms have been mapped using similar techniques. Genes on different chromosomes are not linked to each other and are therefore not amenable to this analysis. The major drawbacks with this type of approach are the requirement for a phenotype for the gene that is being mapped and the number of crosses required to generate accurate mapping data. Additionally, a tacit assumption of mapping based on crosses is that the recombination frequency is equal for all part of the chromosome. This is simply not the case, and many recombinational ‘hot-spots’ and ‘cold-spots’ have been identified. In humans, the segregation of naturally occurring mutant alleles in families can be used to estimate map distances, but the relatively low number of previously identified human genes makes this approach difficult. Several different methods have been used to exploit the inheritance of these variations to map their genomic location. These differences may occur as frequently as about once every 100–300 bp (Collins et al. Some of these alterations will be disease causing mutations – they may change the sequence of amino acids within a protein or alter the way in which gene expression occurs to impair the function of the resulting protein. Some of the nucleotide differences between individuals will, however, result in the alteration of restriction enzyme recognition sites such that existing sites are destroyed or new sites are created (Figure 9. Microsatellites are short, 2–6 bp, tandemly repeated se- quences that occur in a seemingly random fashion distributed throughout the genome of all higher organisms. The number of repeats found at any particular genomic location is highly individual specific. The repeats are thought to be generated by polymerase ‘slippage’ during replication (Schlotterer,¨ 2000). In the first case two small fragments will be formed that are capable of binding the probe, while in the second a single, larger fragment will bind. The restriction fragments are separated on an agarose gel and subjected to Southern blotting (see Figure 2. Dinucleotide microsatellites in mam- mals typically vary in repeat number from about 10 to 30 repeats.

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