Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.

Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle.

To better understand the relationship between the Mr 165,000 M-line protein (M-protein) and H-zone structure in skeletal and in cardiac muscle, as well as the possible interaction of M-protein with another skeletal muscle M-line component, the homodimeric creatine kinase isoenzyme composed of two M subunits (MM-CK), we performed biochemical, immunological, and ultrastructural studies on myofibrils extracted by different procedures. In contrast to MM-CK, M-protein could not be completely removed from myofibrils by low ionic strength extraction. Fab-fragments of antibodies against M-protein could not release M-protein quantitatively from either breast or heart myofibrils but remained bound to the myofibrillar structure, whereas monovalent antibodies against MM-CK cause the specific release of MM-CK and the concomitant disappearance of the M-line from chicken skeletal muscle myofibrils. When MM-CK was removed from skeletal myofibrils by low ionic strength extraction or, more specifically, by incubation with anti-MM-CK Fab, M-protein was still not released quantitatively upon treatment with anti-M-protein Fab as judged from immunofluorescence data. In the ultrastructural investigation of low ionic strength extracted muscle fibers, M protein could be localized in two stripes on both sides of the former M-line, suggesting a reduced attachment to the residual H-zone structure, whereas the specific removal of MM-CK resulted in the same dense staining pattern for M-protein within the M-line as observed in untreated fibers. However, the binding of M-protein to the residual M-line structure seemed to be reduced, as a considerable amount of this protein could be identified in the supernate of sequentially incubated myofibrils. The results indicate a strong binding of M-protein within the H-zone structure of skeletal as well as heart myofibrils.

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross-striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M-line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'-bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.

Change in plasma membrane Ca2(+)-ATPase splice-variant expression in response to a rise in intracellular Ca2+.

BACKGROUND: Most eukaryotic genes are divided into introns and exons. Upon transcription, the intronic segments are eliminated and the exonic sequences spliced together through a series of complex processing events. Alternative splicing refers to the optional inclusion or exclusion of specific exons in transcripts derived from a single gene, which leads to structural and functional changes in the encoded proteins. Although many components of the machinery directing the physical excision of introns and joining of exons have been elucidated in recent years, the signaling pathways regulating the activity of the machinery remain largely unexplored. RESULTS: A calcium-mediated signaling pathway regulates alternative splicing at a specific site of human plasma membrane calcium pump-2 transcripts. This site consists of three exons, which are differentially used in a tissue-specific manner. In IMR32 neuroblastoma cells, a transient elevation of intracellular calcium changed the predominant pattern from one in which all three exons are included to the coexpression of a variant including only the third exon. Western-blot analysis demonstrated that the newly expressed mRNAs are faithfully translated. Once induced, the new splicing pattern was maintained over multiple cell divisions. Protein synthesis was not required to induce the alternative splice change, indicating that all components necessary for a rapid cellular response are present in the cells. CONCLUSIONS: Calcium signaling exerts a direct influence on the regulation of alternative splicing. Notably, a calcium-mediated change in the expression of alternatively spliced variants of a calcium regulatory protein was discovered. The change in splicing occurs quickly, is persistent but reversible and leads to a corresponding change in protein expression. The specific nature in which differently spliced protein variants are expressed, and now the fact that their expression can be regulated by distinct intracellular signaling pathways, suggests that the regulation of alternative splicing by physiological stimuli is a widespread regulatory mechanism by which a cell may coordinate its responses to environmental cues.

Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates.

DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA.

Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene.

To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.

Functional analysis of the promoters of the human CaMIII calmodulin gene and of the intronless gene coding for a calmodulin-like protein.

More than 1 kb of the 5'-flanking DNA of the human CaMIII calmodulin gene and of the calmodulin-like protein (CLP) gene have been sequenced. Notable features are the absence of a TATA-box and the presence of AGGGA elements in both upstream regions, the presence of several sequences with homology to known regulatory elements (cAMP-, retinoic acid- and interferon-responsive elements) in the CLP gene, and a high G + C-content and several putative Sp1-factor-binding sites in the CaMIII gene. 1 kb of the CaMIII upstream region was driving high-level growth hormone (hGH) reporter gene expression in human teratoma and monkey COS cells. Promoter activity dropped to about 30% and 10% when only 252 bp and 114 bp, respectively, of the CaMIII sequence were present. In contrast to the CaMIII promoter, the CLP gene upstream region was driving hGH expression only in human teratoma, but not in monkey COS cells. Addition of retinoic acid to the transfected cells had minimal effects on both promoters, leading to a 10-30% decrease of activity. The results show that the human CaMIII gene contains a strong and ubiquitously active promoter, whereas the promoter of the intronless CLP gene appears to be regulated in a cell-specific manner.

Structural organization of the human CaMIII calmodulin gene.

The complete structural organization of the human calmodulin III gene has been determined. This gene specifies the mRNA represented by the previously reported cDNA ht6. The gene contains six exons spread over a total of approx. 10 kb of DNA. Its exon-intron organization is identical to that of the only known chicken calmodulin gene and to that of two of the three characterized rat calmodulin genes. As in many other genes encoding Ca2+ binding proteins, intron 1 separates the ATG initiation codon from the remainder of the coding region. The major and two minor sites of transcription initiation have been determined by primer extension and ribonuclease protection assays. The DNA sequence in the promoter and 5' untranslated region is extremely GC-rich. No typical TATA and CAAT boxes are present upstream of the major transcriptional start site; however, a consensus CAAT box sequence is found further upstream and may play a role in transcriptional initiation from the minor start sites. Six sequence elements with high similarity to monkey SV40-like Sp1-binding regions are present in the putative promoter region, two of which contain perfect GGGCGG core sequences. The structure of the human calmodulin III gene promoter indicates that this gene belongs to a class of 'house-keeping' genes but that its level of expression may also be specifically regulated.

Minimum CAG repeat in the human calmodulin-1 gene 5' untranslated region is required for full expression.

The human calmodulin-1 gene (hCALM1) contains a (CAG)7 repeat in its 5'-untranslated region (5'-UTR). We found this repeat to be stable and nonpolymorphic in the human population. To determine whether the repeat region affects hCALM1 expression and whether repeat expansions to numbers known to be associated with disease in other genes may alter expression, we tested luciferase reporter genes driven by the hCALM1 promoter and 5'-UTR containing 0, 7 (wild-type), 20, and 45 CAG repeats in human NT2/D1 teratoma cells. Interestingly, the repeat deletion, (CAG)0, decreased expression by 45%, while repeat expansions to (CAG)20 and (CAG)45, or the insertion of a scrambled (C,A,G)7 sequence did not alter gene expression. These data indicate (1) that the endogenous repeat element is required for full expression of hCALM1, and (2) that some triplet repeat expansions in the 5'-UTR of protein-coding genes may be well tolerated and even optimize gene expression.

Primary structure of human plasma membrane Ca(2+)-ATPase isoform 3.

The complete coding sequence of the human plasma membrane calcium ATPase (PMCA) isoform 3 was determined from overlapping genomic and cDNA clones. The cDNAs for the two major alternative splice variants 3a (3CII) and 3b (3CI) code for proteins of 1173 and 1220 amino-acid residues, respectively, which show 98% identity with the corresponding rat isoforms. On a multiple human tissue Northern blot, a major PMCA3 transcript of about 7 kb was detected exclusively in the brain, demonstrating the highly restricted pattern of expression of this isoform to human neuronal tissues. With the elucidation of the human PMCA3 primary structure, complete sequence information is now available for the entire family of human PMCA isoforms.

Ultrastructural localization of M-band proteins in chicken breast muscle as revealed by combined immunocytochemistry and ultramicrotomy.

Cryo-ultramicrotomy and "conventional" plastic sectioning have been used in combination with extraction and immunolabeling techniques to determine the location of the two M-band proteins characterized to date, MM-creatine kinase (MM-CK: Mr, 80,000) and M-protein "myomesin" (Mr, 165,000) within the M-region of chicken pectoralis muscle. The following main results were obtained. (1) The M-band in chicken pectoralis muscle contains five major striations (M1, M4 and M4', M6 and M6' in the terminology of Sjöström & Squire, 1977a). (2) Extraction of the bulk of the electron-dense M-band with low ionic strength removes the M-striations M1, M4 and M4' while M6 and M6' are retained. Cross-sections through the M-region of such muscles lack primary M-bridges connecting the thick myosin filaments. (3) Labeling with antibodies against MM-CK enhances the M-striations M4 and M4'; sometimes the whole region between M4 and M4' is labeled. (4) Incubation with antibodies against myomesin results in the labeling of the whole M-band from M6 to M6'; no label is found in the rest of the bare zone outside M6 and M6'. (5) Incubation of low ionic strength extracted muscle fibers with antibodies against myomesin leads to an "incomplete" labeling of the M-band between M6 and M6'; lines M6 and M6' are sometimes seen to be enhanced presumably due to antibody labeling. From these results it is concluded that MM-CK is the major protein of the M4 and M4' (and possibly also of the M1) M-bridges. Myomesin is bound within the M-band along the thick filaments from M6 to M6'. Two hypothetical models for the possible location of myomesin are discussed. According to these models myomesin would either make up the M-filaments or be directly attached to and along the central bare zone of thick myosin filaments.

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod.

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence. The rat embryonic MHC polypeptide is encoded by exons 3 to 41 and contains 1939 amino acid residues with a calculated Mr of 223,900. Its amino acid sequence displays the structural features typical for all sarcomeric MHCs, i.e. an amino-terminal "globular" head region and a carboxy-terminal alpha-helical rod portion that shows the characteristics of a coiled coil with a superimposed 28-residue repeat pattern interrupted at only four positions by "skip" residues. The complex structure of the rat embryonic MHC gene and the conservation of intron locations in this and other MHC genes are indicative of a highly split ancestral sarcomeric MHC gene. Introns in the rat embryonic gene interrupt the coding sequence at the boundaries separating the proteolytic subfragments of the head, but not at the head/rod junction or between the 28-residue repeats present within the rod. Therefore, there is little evidence for exon shuffling and intron-dependent evolution by gene duplication as a mechanism for the generation of the ancestral MHC gene. Rather, intron insertion into a previously non-split ancestral MHC rod gene consisting of multiple tandemly arranged 28-residue-encoding repeats, or convergent evolution of an originally non-repetitive ancestral MHC rod gene must account for the observed structure of the rod-encoding portion of present-day MHC genes.

The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools?

Calmodulin (CaM) is a ubiquitous, highly conserved calcium sensor protein involved in the regulation of a wide variety of cellular events. In vertebrates, an identical CaM protein is encoded by a family of non-allelic genes, raising questions concerning the evolutionary pressure responsible for the maintenance of this apparently redundant family. Here we review the evidence that the control of the spatial and temporal availability of CaM may require multiple regulatory levels to ensure the proper localization, maintenance and size of intracellular CaM pools. Differential transcription of the CaM genes provides one level of regulation to meet tissue-specific, developmental and cell-specific needs for altered CaM levels. Post-transcriptional regulation occurs at the level of mRNA stability, perhaps dependent on alternative polyadenylation and differences in the untranslated sequences of the multiple gene transcripts. Recent evidence indicates that trafficking of specific CaM mRNAs may occur to specialized cellular locales such as the dendrites of neurons. This could allow local CaM synthesis and thereby help generate local pools of CaM. Local CaM activity may be further regulated by post-translational mechanisms such as phosphorylation or storage of CaM in a 'masked' form. The spatial resolution of CaM activity is enhanced by the limited free diffusion of CaM combined with differential affinity for and availability of target proteins. Preserving multiple CaM genes with divergent noncoding sequences may be necessary in complex organisms to ensure that the many CaM-dependent processes occur with the requisite spatial and temporal resolution. Transgenic mouse models and studies on mice carrying single and double gene 'knockouts' promise to shed further light on the role of specificity versus redundancy in the evolutionary maintenance of the vertebrate CaM multigene family.

Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3.

Human calmodulin is encoded by three genes CALM1, CALM2 and CALM3 located on different chromosomes. To complete the characterization of this family, the exon-intron structure of CALM2 was solved by a combination of genomic DNA library screening and genomic PCR amplification. Intron interruptions were found at identical positions in human CALM2 as in CALM1 and CALM3; however, the overall size of CALM2 (16 kb) was almost twice that of the other two human CALM genes. Over 1 kb of the 5' flanking sequence of human CALM2 were determined, revealing the presence of a TATA-like sequence 27 nucleotides upstream of the transcriptional start site and several conserved sequence elements possibly involved in the regulation of this gene. To determine if differential transcriptional activity plays a major role in regulating cellular calmodulin levels, we directly measured and compared the mRNA abundance and transcriptional activity of the three CALM genes in proliferating human teratoma cells. CALM3 was at least 5-fold more actively transcribed than CALM1 or CALM2. CALM transcriptional activity agreed well with the mRNA abundance profile in the teratoma cells. In transient transfections using luciferase reporter genes driven by 1 kb of the 5' flanking DNA of the three CALM genes, the promoter activity correlated with the endogenous CALM transcriptional activity, but only when the 5' untranslated regions were included in the constructs. We conclude that the CALM gene family is differentially active at the transcriptional level in teratoma cells and that the 5' untranslated regions are necessary to recover full promoter activation.

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy.

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.

Differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart.

The differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart has been investigated. Several aspects of some of the structural proteins, that means the intermediate filament subunit skeletin, myosin, and the myofibrillar M-line proteins MM-creatine kinase and myomesin have been studied by using gel electrophoresis as well as enzyme and immunohistochemical techniques in combination with electron microscopy of both plastic and cryosectional material. We show that marked changes take place in the organization of the intermediate filament system and in the contractile apparatus, both in atria and in ventricles of the rat heart during postnatal development. In the newborn rats no dense myofibrillar M-bands were present in the M-region and the sarcomeric bands were irregular while in the four-week-old rats dense M-bands composed of a set of five crossbridges interconnecting the thick filaments were present. The sarcomeric bands were now regular. These observations are related to the presence of different isomyosins in the atria and in the ventricles of the newborn and the four-week-old rats, to the observation that MM-creatine kinase was only present in the M-region in the four-week old rats and to the physiological maturation of the heart.

Loss of immunoreactivity for human calmodulin-like protein is an early event in breast cancer development.

Cell proliferation requires calmodulin, a protein that regulates calcium-dependent enzymes involved in signal transduction pathways in eukaryotic cells. Calmodulin-like protein (CLP) is found in certain epithelial cell types, including normal breast epithelium, and, although it closely resembles calmodulin in amino acid sequence, CLP interacts with different proteins than does calmodulin. The observation that CLP mRNA expression is dramatically reduced in transformed breast epithelial cells led to two hypotheses: (1) CLP helps to maintain the differentiated state in epithelial cells; and (2) downregulation of CLP accompanies malignant transformation of breast epithelial cells. The objective of this study was to determine if the expression of CLP in human breast cancer specimens is reduced in comparison to its expression in normal breast tissue. Eighty human breast cancer biopsy specimens were analyzed immunohistochemically for CLP expression by using a polyclonal rabbit antihuman CLP antibody. CLP expression was reduced in 79% to 88% of the invasive ductal carcinoma and lobular carcinoma specimens and in a similar fraction of the ductal carcinoma in-situ specimens, compared with normal breast specimens. None of the breast cancer specimens showed an increase in CLP expression. These findings support the hypotheses that CLP behaves as a functional tumor suppressor protein and is downregulated early in breast cancer progression.

Cloning an Escherichia coli gene encoding a protein remarkably similar to mammalian aldehyde dehydrogenases.

The nucleotide (nt) sequence of 2.9 kb of Escherichia coli genomic DNA that encompasses a gene encoding a putative aldehyde dehydrogenase (ALDH) has been determined. The presence of an open reading frame beginning 2 nt downstream from the ALDH-coding sequence indicates that this gene may be part of a larger operon. An extended upstream nt sequence displays striking similarity to sequences found upstream or in intergenic regions of a number of other bacterial genes. Crude cell extracts from E. coli grown under several different conditions show weak but measurable ALDH enzyme activity that prefers NADP+ over NAD+ as coenzyme; however, aldH gene expression appears to be very low, since no specific transcripts derived from the novel gene can be detected on Northern blots of RNA isolated from these cells. The deduced E. coli protein contains 495 amino acid (aa) residues with a calculated Mr of 53418. Its aa sequence showed marked similarity to NAD(+)-dependent ALDHs of eukaryotes. About 40% aa sequence identity, and over 60% similarity, are detected between the E. coli protein and both the cytosolic (class-1) and the mitochondrial (class-2) forms of mammalian ALDHs. In contrast to the mammalian enzymes, which contain eight to eleven Cys residues, only four Cys are present in the E. coli protein, and of these only Cys302, corresponding to the disulfiram-sensitive residue in the mammalian enzymes, is found at a conserved position in both the prokaryotic and the eukaryotic ALDHs. The availability of a bacterial ALDH with a high degree of similarity to mammalian ALDHs promises to facilitate future structural studies on these enzymes.

Characterization of two novel human retropseudogenes related to the calmodulin-encoding gene, CaMII.

Two human genomic clones (lambda hg22 and lambda hg29), containing two novel calmodulin (CaM) retropseudogenes, were isolated and characterized. The two pseudogenes show high similarity with the human CaMII cDNA, hCE1 [SenGupta et al., J. Biol. Chem. 262 (1987) 16663-16670] and the CaMII-type retropseudogene, hCE2 (CaMII-psi 1) [SenGupta et al., Nucleic Acids Res. 17 (1989) 2868]. One of them, in clone lambda hg22 (CaMII-psi 2), shows all the characteristics of a processed pseudogene. In clone lambda hg29 (CaMII-psi 3), however, an Alu repetitive sequence was detected immediately upstream from the ancestral 5'-untranslated region. Downstream from the truncated 3'-untranslated region, three additional copies of Alu repetitive sequences flanking about 750 nucleotides of unknown origin were found. Such a processed retropseudogene flanked by multiple Alu repeats may be a target for further recombination events. The three retropseudogenes CaMII-psi 1, CaMII-psi 2 and CaMII-psi 3 are estimated to be about 49, 21 and 25 million years old, respectively.

Characterization of an intronless human calmodulin-like pseudogene.

We report the isolation and characterization of a human genomic clone encoding a calmodulin-like pseudogene. It contains an open reading frame of 444 nucleotides, not interrupted by introns. The nucleotide sequence of the open reading frame shows 80%, 71% and 69% identity to the previously reported human calmodulin cDNAs lambda ht6 [17], hCWP [22], and lambda hCE1 [23], respectively. The derived amino acid sequence has only 85% identity to vertebrate calmodulin, but shows four potentially functional Ca2+-binding loops. In the human tissues tested, this pseudogene is not expressed, though gene structure including promoter elements and a putative polyadenylation site seems to be intact.